System and Methods for Massively Parallel Analysis of Nucleic Acids in Single Cells

ABSTRACT

Methods and systems are provided for massively parallel genetic analysis of single cells in emulsion droplets or reaction containers. Genetic loci of interest are targeted in a single cell using a set of probes, and a fusion complex is formed by molecular linkage and amplification techniques. Methods are provided for high-throughput, massively parallel analysis of the fusion complex in a single cell in a population of at least 10,000 cells. Also provided are methods for tracing genetic information back to a cell using barcode sequences.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/993,047, filed Jun. 10, 2013, which is a national stage entry ofPCT/US2011/065600, filed Dec. 16, 2011, which claims the benefit of U.S.Provisional Application No. 61/459,600, filed Dec. 16, 2010, thedisclosures of which are hereby incorporated by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 19, 2016, isnamed 33974_US_CRF_Sequencelisting.txt and is 17,383 bytes in size.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the fields of molecular biology and moleculardiagnostics, and more specifically to methods for massively parallelgenetic analysis of nucleic acids in single cells.

2. Description of the Related Art

Certain quantitative genetic analyses of biological tissues andorganisms are best performed at the single cell level. However, singlecells only contain picograms of genetic material. Conventional methods,such as polymerase chain reaction (PCR), RNA sequencing (Mortazavi etal., 2008 Nature Methods 5:621-8), chromatin immunoprecipitationsequencing (Johnson et al., 2007 Science 316:1497-502), or whole genomesequencing (Lander et al., 2001 Nature 409:860-921), require moregenetic material than is found in a single cell and are usuallyperformed with thousands to millions of cells. These techniques provideuseful genetic information at the cell population level, but haveserious limitations for understanding biology at the single cell level.Current biological tools also lack the capacity to assay geneticmeasurements in many single cells in parallel.

Conventional single cell techniques are slow, tedious, and limited inthe quantity of cells that can be analyzed at once. For example, inpre-implantation genetic diagnosis (PGD), a single cell is removed froma cleavage stage human embryo for genome-wide analysis of geneticdiseases (Johnson et al., 2010 Human Reproduction 25:1066-75).Applications such as PGD require time-consuming, hand-guided biopsytechnology, and the largest studies include hundreds of single cells. Inanother example, genetic recombination between loci of interest can bemeasured in single sperm cells (Jiang et al., 2005 Nucleic AcidsResearch 33:e91), but a manual analysis of thousands of single spermwould be time-consuming and impractical.

An established method for single cell analysis is fluorescence-activatedcell sorting (FACS). Single cells are diluted into reaction wells, andvarious genetic and molecular biology techniques can be performed in thewells, from whole genome amplification to single locus PCR assays.However, due to the physical limits of parallelization using reactionwells, FACS is only useful for analyzing hundreds of single cells,rather than hundreds of thousands of single cells.

Single cells can also be used as reaction compartments for performingvarious genetic analyses (Embleton et al., 1992 Nucleic Acids Research20:3831-37; Hviid, 2002 Clinical Chemistry 48:2115-2123; U.S. Pat. No.5,830,663). Single cells can be sorted in aqueous-in-oil microdropletemulsions, and molecular analyses can be performed in the microdroplets(Johnston et al., 1996 Science 271:624-626; Brouzes et al., 2009 PNAS106:14195-200; Kliss et al., 2008 Anal Chem 80:8975-81; Zeng et al.,2010 Anal Chem 82:3183-90). These single cell assays are limited tosingle cell PCR in emulsions, or in situ PCR in single fixed andpermeabilized cells. Moreover, when analyzing large populations ofcells, it is difficult to trace back each gene product to a single cellor subpopulations of cells.

Thus, there is a need for methods for high-throughput, massivelyparallel genetic characterization of single cells and methods foridentifying the cell or subpopulation of cells that originated thegenetic material.

SUMMARY OF THE INVENTION

Disclosed herein is a method for analyzing at least two nucleic acidsequences in a single cell contained within a population of at least10,000 cells. The method includes providing a providing a first set ofnucleic acid probes, the first set comprising a first probe comprising asequence that is complementary to a first target nucleic acidsubsequence, a second probe comprising a sequence that is complementaryto a second subsequence of the first target nucleic acid and a secondsequence that is complementary to an exogenous sequence, a third probecomprising the exogenous sequence and a sequence that is complementaryto a first subsequence of a second target nucleic acid, and a fourthprobe comprising a sequence that is complementary to a secondsubsequence of the second target nucleic acid sequence.

The method includes isolating the single cells with at least one set ofnucleic acid probes; amplifying the first and second target nucleic acidsequences independently, wherein the first target nucleic acid sequenceis amplified using the first probe and the second probe, and wherein thesecond target nucleic acid sequence is amplified using the third probeand the fourth probe; hybridizing the exogenous sequence to itscomplement; and amplifying the first target nucleic acid sequence, thesecond target nucleic acid sequence, and the exogenous sequence usingthe first and fourth probes, thereby generating a fused complex.

The method also provides performing a bulk sequencing reaction togenerate sequence information for at least 100,000 fused complexes fromat least 10,000 cells within the population of cells, wherein thesequence information is sufficient to co-localize the first targetnucleic acid sequence and the second target nucleic acid sequence to asingle cell from the population of at least 10,000 cells.

In one aspect, the single cell is isolated in an emulsion microdroplet.In another aspect, the single cell is isolated in a reaction container.

In one embodiment, the amplifying step includes performing a polymerasechain reaction, wherein the amplifying step comprises performing apolymerase chain reaction, and wherein the first and third probes areforward primers and the second and fourth probes are reverse primers forthe polymerase chain reaction. In another embodiment, the amplifyingstep includes performing a polymerase chain reaction, wherein the firstand third amplification primers are forward primers and the second andfourth amplification primers are reverse primers for the polymerasechain reaction. In some embodiments, the amplifying step comprisesperforming a ligase chain reaction. The amplifying step can includeperforming a polymerase chain reaction, a reverse-transcriptasepolymerase chain reaction, a ligase chain reaction, or a ligase chainreaction followed by a polymerase chain reaction.

In another embodiment, the fused complex is circular. In someembodiments, the first or second target nucleic acid sequence is an RNAsequence. The first or second target nucleic acid sequence can also be aDNA sequence. In one embodiment, the first or second target nucleic acidsequence comprises a T-cell receptor sequence. In another embodiment,the first target nucleic sequence, the second target nucleic acidsequence or both target nucleic acid sequences comprises animmunoglobulin sequence. In other embodiments, the first target nucleicacid comprises a T-cell receptor sequence, and the second target nucleicsequence comprises a second molecule that is associated with immune cellfunction. In another embodiment, the first target nucleic acid sequencecomprises an immunoglobulin sequence, and the second sequence comprisesa second molecule associated with immune cell function. In oneembodiment, the second molecule associated with immune cell function isselected from the group consisting of: interleukin-2 (IL-2),interleukin-4 (IL-4), interferon gamma (IFNγ), interleukin-10 (IL-10),interleukin-1 (IL-1), interleukin-13 (IL-13), interleukin-17 (IL-17),interleukin-18 (IL-18), tumor necrosis factor alpha (TNFα), tumornecrosis factor beta (TNFβ), T-box transcription factor 21 (TBX21),forkhead box P3 (FOXP3), cluster of differentiation 4 (CD4), cluster ofdifferentiation 8 (CD8), cluster of differentiation 1d (CD1d), clusterof differentiation 161 (CD161), cluster of differentiation 3 (CD3),major histocompatibility complex (MHC), cluster of differentiation 19(CD19), interleukin 7 receptor (IL-17 receptor), cluster ofdifferentiation 10 (CD10), cluster of differentiation 20 (CD20), clusterof differentiation 22 (CD22), cluster of differentiation 34 (CD34),cluster of differentiation 27 (CD27), cluster of differentiation 5(CD5), and cluster of differentiation 45 (CD45), cluster ofdifferentiation 38 (CD38), cluster of differentiation 78 (CD78),interleukin-6 receptor, Interferon regulatory factor 4 (IRF4), clusterof differentiation 138 (CD138).

In an embodiment, the first or second target nucleic acid includes arare gene sequence. In some embodiments, the rare gene sequence ispresent in fewer than 5% of the cells, fewer than 1% of the cells, orfewer than 0.1% of the cells. In one embodiment, the rare gene sequenceresults from a genetic mutation. In another embodiment, the geneticmutation is a somatic mutation. In an embodiment, the genetic mutationis a mutation in a gene selected from the group consisting of epidermalgrowth factor receptor (EGFR), phosphatase and tensin homolog (PTEN),tumor protein 53 (p53), MutS homolog 2 (MSH2), multiple endocrineneoplasia 1 (MEN1), adenomatous polyposis coli (APC), Fas receptor(FASR), retinoblastoma protein (Rb1), Janus kinase 2 (JAK2), (ETS)-liketranscription factor 1 (ELK1), v-ets avian erythroblastosis virus E26oncogene homolog 1 (ETS1), breast cancer 1 (BRCA1), breast cancer 2(BRCA2), hepatocyte growth factor receptor (MET), ret protoco-oncogene(RET), V-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (HER2),V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), B-celllymphoma 2 (BCL2), V-myc myelocytomatosis viral oncogene homolog (MYC),neurofibromatosis type 2 gene (NF2), v-myb myeloblastosis viral oncogenehomolog (MYB), and mutS homolog 6 (E. coli) (MSH6). In otherembodiments, the mutation is associated with a disease, and in oneembodiment, the disease is cancer. In some embodiments, the cancer is acancer selected from the group consisting of lung carcinoma, non-smallcell lung cancer, small cell lung cancer, uterine cancer, thyroidcancer, breast carcinoma, prostate carcinoma, pancreas carcinoma, coloncarcinoma, lymphoma, Burkitt lymphoma, Hodgkin lymphoma, myeloidleukemia, leukemia, sarcoma, blastoma, melanoma, seminoma, brain cancer,glioma, glioblastoma, cerebellar astrocytoma, cutaneous T-cell lymphoma,gastric cancer, liver cancer, ependymona, laryngeal cancer, neck cancer,stomach cancer, kidney cancer, pancreatic cancer, bladder cancer,esophageal cancer, testicular cancer, medulloblastoma, vaginal cancer,ovarian cancer, cervical cancer, basal cell carcinoma, pituitaryadenoma, rhabdomyosarcoma, or Kaposi sarcoma.

In addition, the method can include in certain embodiments fixing andpermeabilizing the cells prior to performing the amplification step. Themethod can also include lysing the cells prior to performing theamplification step and quantifying the sequence information generatedfrom the bulk sequencing reaction.

In some embodiments, the method for analyzing the single cell includes asingle cell contained within a population of at least 25,000 cells, atleast 50,000 cells, at least 75,000 cells, or at least 100,000 cells. Insome embodiments, the single cell is a unique cell with respect to theremaining cells in the population. In other embodiments, the single cellis a representative of a subpopulation of cells within the population.The population can be considered in some embodiments to be the totalnumber of cells analyzed in a method of the invention.

In one embodiment, performing the bulk sequencing reaction to generatesequence information is carried out for at least 1,000,000 fusedcomplexes from at least 10,000 cells within the population of cells.

The method includes providing a second set of nucleic acid probes, thesecond set comprising a fifth probe comprising a sequence that iscomplementary to a third target nucleic acid subsequence, a sixth probecomprising a sequence that is complementary to a second subsequence ofthe third target nucleic acid sequence and a second sequence that iscomplementary to a second exogenous sequence, a seventh probe comprisingthe exogenous sequence and a sequence that is complementary to a firstsubsequence of a fourth target nucleic acid sequence, and an eighthprobe comprising a sequence that is complementary to a secondsubsequence of the fourth target nucleic acid sequence.

The method also provides isolating the single cells with the first andsecond sets of nucleic acid probes; amplifying the third and fourthtarget nucleic acid sequences independently, wherein the third targetnucleic acid sequence is amplified using the fifth probe and the sixthprobe, and wherein the fourth target nucleic acid sequence is amplifiedusing the seventh probe and the eighth probe; hybridizing the exogenoussequence to its complement; amplifying the third target nucleic acidsequence, the fourth target nucleic acid sequence and the exogenoussequence using the fifth and eighth probes, thereby generating a fusedcomplex; and performing a bulk sequencing reaction to generate sequenceinformation for at least 100,000 fused complexes from at least 10,000cells within the population of cells, wherein the sequence informationis sufficient to co-localize the first target nucleic acid sequence, thesecond target nucleic acid sequence, the third target nucleic acidsequence, and the fourth target nucleic acid sequence to a single cellfrom the population of at least 10,000 cells.

In some aspects, the first target nucleic acid sequence and the thirdtarget nucleic acid sequence are the same. In other aspects, the firsttarget nucleic acid sequence and the third target nucleic acid sequenceare different.

In other embodiments, the method includes providing N sets of nucleicacid probes, wherein each of the N sets comprise an I₁ probe comprisinga sequence that is complementary to an I_(a) target nucleic acid firstsubsequence, an I₂ probe comprising a sequence that is complementary toan I_(a) target nucleic acid second subsequence and a second sequencethat is complementary to an I exogenous sequence, an I₃ probe comprisingthe I exogenous sequence and a sequence that is complementary to anI_(b) target nucleic acid first subsequence, and an I₄ probe comprisinga sequence that is complementary to an I_(b) target nucleic acid secondsubsequence, wherein I ranges from 1 to N

The method also includes isolating the single cells with the N sets ofnucleic acid probes; amplifying for all values of I, the I_(a) and I_(b)target nucleic acid sequences independently, wherein the I_(a) targetnucleic acid sequence is amplified using the I₁ probe and the I₂ probeand the I_(b) target nucleic acid sequence is amplified using the I₃probe and the I₄ probe; hybridizing the I exogenous sequence to itscomplement; amplifying for each I, the I_(a) target sequence, the I_(b)target sequence and the I exogenous sequence using the I₁ and I₄ probes,thereby generating N fused complexes; and performing a bulk sequencingreaction to generate sequence information for at least 100,000 fusedcomplexes from at least 10,000 cells within the population of cells,wherein the sequence information is sufficient to co-localize the NI_(a) target nucleic acid sequence and the I_(b) target nucleic acidsequence to a single cell from the population of at least 10,000 cells.

In other embodiments, N is less than or equal to 10, less than or equalto 100, less than or equal to 1000, less than or equal to 10,000, lessthan or equal to 100,000 or N represents all of the polyadenylatedtranscripts in a cell.

In some embodiments the method includes introducing a unique barcodesequence comprising at least six nucleotides into each of the pluralityof single cells, wherein each barcode sequence is selected from a poolof barcode sequences with greater than 1000-fold diversity in sequences.For each of the plurality of single cells, the method includes providingat least one set of nucleic acid probes. The method includes steps foranalyzing at least two nucleic acid sequences in a single cell containedwithin a population of at least 10,000 cells, comprising isolating eachof a plurality of single cells from a population of at least 10,000cells in an emulsion microdroplet or a reaction container. The methodincludes introducing a unique barcode sequence comprising at least sixnucleotides into each of the plurality of single cells, wherein eachbarcode sequence is selected from a pool of barcode sequences withgreater than 1000-fold diversity in sequence.

For each of the plurality of single cells, the method provides at leastone set of nucleic acid probes, the set comprising a first probecomprising a sequence that is complementary to a nucleic acid sequencethat is located at the 5′ end of the barcode sequence, a second probecomprising a sequence that is complementary to a nucleic acid sequencethat is located at the 3′ end of the barcode sequence and a secondregion of sequence that is complementary to a non-human, exogenoussequence, a third probe comprising a sequence that comprises thenon-human, exogenous sequence and a sequence that is complementary to afirst subsequence of a second target nucleic acid sequence, and a fourthprobe comprising a sequence that is complementary to a secondsubsequence of the second target nucleic acid sequence.

The method continues by amplifying the first and second nucleic acidsequences independently, wherein the first target nucleic acid sequenceis amplified using the first probe and the second probe, and wherein thesecond target nucleic acid sequence is amplified using the third probeand the fourth probe; hybridizing the exogenous sequence to itscomplement; amplifying the first target nucleic acid sequence, thesecond target nucleic acid sequence, and the exogenous sequence usingthe first and fourth probes; performing bulk sequencing of the fusedcomplexes; and identifying a single cell for each of the fused complexesbased on the barcode sequence.

In some embodiments, the barcode sequence is affixed to a bead or asolid surface. The bead or the solid surface can be isolated in theemulsion microdroplet or the reaction container.

In other embodiments, the method includes introducing a unique barcodesequence comprises fusing the emulsion microdroplet or a reactioncontainer comprising the single cell with the emulsion microdroplet or areaction container comprising the barcode sequence affixed to the beador the solid surface. The second target nucleic acid sequence can becomplementary to an RNA sequence. The second target nucleic acidsequence can be complementary to a DNA sequence.

In certain embodiments, amplifying comprises performing a polymerasechain reaction, performing a ligase chain reaction, or performing byligase chain reaction followed by polymerase chain reaction.

In one embodiment, the single cell is contained within a population ofat least 25,000 cells. In other embodiments, the single cell iscontained within a population of at least 50,000 cells. The single cellcan be contained within a population of at least 75,000 cells or withina population of at least 100,000 cells.

In certain embodiments, the method also includes quantifying the fusedcomplexes. In other embodiments, the fused complexes are circular.

In one aspect, the method includes providing N sets of nucleic acidprobes, wherein each of the N sets comprise an I₁ probe comprising asequence that is complementary a first subsequence of a barcodesequence, an I₂ probe comprising a sequence that is complementary to asecond subsequence of the barcode sequence and a second sequence that iscomplementary to an I exogenous sequence, an I₃ probe comprising the Iexogenous sequence and a sequence that is complementary to an I_(b)target nucleic acid first subsequence, and an I₄ probe comprising asequence that is complementary to an I_(b) target nucleic acid secondsubsequence, wherein I ranges from 1 to N.

The method also provides isolating the single cells with the N sets ofnucleic acid probes; amplifying for all values of I, the barcodesequence and the I_(b) target nucleic acid sequences independently,wherein the barcode sequence is amplified using the I₁ probe and the I₂probe and the I_(b) target nucleic acid sequence is amplified using theI₃ probe and the I₄ probe; hybridizing the I exogenous sequence to itscomplement; amplifying for each I, the barcode sequence, the I_(b)target sequence and the I exogenous sequence using the I₁ and I₄ probes,thereby generating N fused complexes; and performing a bulk sequencingreaction to generate sequence information for at least 100,000 fusedcomplexes from at least 10,000 cells within the population of cells,wherein the sequence information is sufficient to co-localize thebarcode sequence and the I_(b) target nucleic acid sequence to a singlecell from the population of at least 10,000 cells.

In other aspects, N is less than or equal to 10, less than or equal to100, less than or equal to 1000, less than or equal to 100,000, or Nrepresents all of the polyadenylated transcripts in a cell. In someembodiments, the barcode sequence is the same sequence for all N.

In other embodiments the invention also provides a method forintroducing said barcode sequences into reaction containers or emulsionmicrodroplets. The method includes providing a pool of unique barcodesequences, wherein each barcode sequence is linked to a selectionresistance gene. The method also includes providing a population ofsingle cells, transfecting the population of single cells with the poolof unique barcode sequences, selecting cells containing a unique barcodesequence and the selection resistance gene, and isolating each of theselected cells into reaction containers or emulsion microdroplets. Theselection resistance gene can encode resistance to gentamycin, neomycin,hygromycin, or puromycin, for example.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, and accompanying drawings, where:

FIG. 1A shows an example of sequence linkage in a single cell byintra-cellular multiprobe circularization of a molecular complex,according to one embodiment of the invention. Each probe has a region ofcomplementarity to each of the target loci. The complex includes twonucleic acid probes (a and b) and two target nucleic acids (c and d).The single cell (e) can be contained in a reaction container or anemulsion droplet (j).

FIG. 1B illustrates an example of sequence linkage in a single cell(also in a reaction container or emulsion droplet (j)) by intra-cellularmultiprobe circularization of a complex, according to one embodiment ofthe invention. The two nucleic acid probes (a and b) are hybridized tothe complementary regions of the two target nucleic acids (c and d).

FIG. 1C illustrates an example of circularization of a probe-targetlinkage complex occurs by amplification, according to one embodiment ofthe invention.

FIG. 2 is an example of amplification of a circularized probe-targetlinkage complex (a) using a polymerase (b), according to one embodimentof the invention. In some embodiments, a φ-29 polymerase is used in amediated rolling circle amplification, and copies (b and c) of thecircularized probe-target complex are generated.

FIG. 3 illustrates an example of amplification of a circularizedprobe-target linkage complex (a) using a polymerase (b) and primers (cand d), according to one embodiment of the invention. The primers (c andd) are used to amplify the region of the circularized probe-targetcomplex that is complementary to the target nucleic acid. Multiplecopies (e) of a linear double-stranded polynucleic acid amplicon aregenerated and sequenced in bulk.

FIG. 4 illustrates an example of amplification of a circularizedprobe-target linkage complex (a) in a single cell (b), according to oneembodiment of the invention Amplification occurs by transformation intobacteria and subsequent selection with antibiotics. The amplicon (a)contains an antibiotic resistant gene and cells (c) that are transformedwith the amplicon are selected in the presence of antibiotics. Cellswithout the circularized probe-target complex (d) are not selected.

FIG. 5A shows an example of single cell sequence linkage byintracellular overlap extension polymerase chain reaction, according toone embodiment of the invention. A forward primer (a) targets one locusof a first target nucleic acid (g). A reverse primer (b) targets anotherlocus of the first target nucleic acid (g) and has a region ofcomplementarity (c) to a region (d) of the forward primer (e). Theforward primer (e) has a region of complementarity to the second targetnucleic acid (h) and the reverse primer (f) targets another region ofthe second target nucleic acid (h). The steps of FIG. 5 can be performedin a reaction container or an emulsion droplet.

FIG. 5B illustrates an example of the hybridization of the probes (a, b,e and f) to respective target nucleic acids (g and h), according to oneembodiment of the invention.

FIG. 6A illustrates an example of the complementary regions (c) and (d)between amplicons (g) and (h), according to one embodiment of theinvention. FIG. 6B shows linkage amplification of the amplicons (g) and(h) using polymerase (e) to create a linked major amplicon (i). The endproduct is a library of “major amplicons” that include the linkedamplicons (g) and (h), which can be sequenced in bulk. The steps of FIG.6 can be performed in a reaction container or an emulsion droplet.

FIGS. 7A and 7B illustrate an example of single cell sequence linkage byintracellular ligase chain reaction combined with overlap extensionpolymerase chain reaction, according to one embodiment of the invention.

FIG. 8A shows an example of the complementary regions between amplicons(a) and (d), according to one embodiment of the invention. FIG. 8B showslinkage amplification of the amplicons using polymerase (e) to create alinked major amplicon. The steps of FIGS. 7 and 8 can be performed in areaction container or an emulsion droplet.

FIG. 9A shows an example of a linked amplicon (f), according to oneembodiment of the invention. FIG. 9B shows the resulting ampliconproduced from the steps shown in FIGS. 8A and 8B. The end product can bea library of “major amplicons” and are be sequenced in bulk.

FIG. 10 illustrates an example of the components required for a singlecell sequence linkage by padlock probes combined with overlap extensionpolymerase chain reaction, according to one embodiment of the invention.

FIG. 11 shows the complementary regions between a first padlock probe(a) and the first target nucleic acid (c) and between a second padlockprobe (b) and a second target nucleic acid (d) in a single cell,according to one embodiment of the invention.

FIG. 12 illustrates the resulting circularized amplicons (g) and (h) andthe primers that are used to amplify the circularized amplicons,according to one embodiment of the invention.

FIG. 13 shows an example of the resulting amplicons from amplificationof the circular probes (g) and (h), according to one embodiment of theinvention.

FIG. 14 shows an example of overlap extension PCR amplification of theamplicons using a polymerase (e), according to one embodiment of theinvention.

FIG. 15 illustrates an example of plasmid library deconvolution bybarcoded tailed end (5′-end barcoded) polymerase chain reaction, whichis followed by bulk sequencing and informatics, according to oneembodiment of the invention. The barcode sequence can be traced back toa well and plate position, the barcode sequence can then be traced to anucleic acid sequence, and the nucleic acid sequence is traced back to awell. Each of the primers in (a) and (b) have a 5′-end barcoded tag. Thetarget nucleic acids in (c) and (d) are amplified using the primers in(a) and (b). The steps can be performed in enclosed containers oremulsion droplets, as shown in (c) and (d).

FIG. 16 shows an example of amplification (e, f) of two target nucleicacids (A and B) using primers that include barcode sequences, accordingto one embodiment of the invention. The resulting amplicons that includethe barcode sequences are shown in (g) and (h).

FIG. 17 shows a simplified example of tracing back a barcode sequence inan amplicon to a cell target (A or B), and tracing back the cell targetto a physical location (c, d) (e.g., a well), according to oneembodiment of the invention.

FIG. 18 illustrates molecular linkage between two transcripts (g and h)and a molecular barcode sequence (k), according to one embodiment of theinvention.

FIG. 19 shows an example of amplification of the target nucleic acids (gand h) using primers as shown, according to one embodiment of theinvention.

FIG. 20 shows an example of amplicons resulting after amplification oftwo target nucleic acids and a barcode sequence (k), according to oneembodiment of the invention.

FIG. 21 illustrates a fused amplicon that includes sequences of twotarget nucleic acids (g and h) and a barcode sequence (k) inside anemulsion droplet or reaction container (j), according to one embodimentof the invention. The fused (“major”) amplicon can be isolated byreverse emulsion and bulk sequenced.

FIG. 22 is an example of molecular linkage between two transcripts (gand h) and a molecular barcode sequence (k) attached to a bead (m),according to one embodiment of the invention.

FIG. 23 illustrates the forward and reverse primers that are used in amolecular linkage between two transcripts (g and h) and a molecularbarcode sequence (k) attached to a bead (m), according to one embodimentof the invention.

FIG. 24 shows an example of amplicons resulting after amplification oftwo target nucleic acids and a barcode sequence (k) attached to a bead(m), according to one embodiment of the invention.

FIG. 25 illustrates a fused amplicon that includes sequences of twotarget nucleic acids (g and h) and a barcode sequence (k), inside anemulsion droplet or reaction container (j), according to one embodimentof the invention. The fused (“major”) amplicon can be isolated byreverse emulsion and bulk sequenced.

FIG. 26 is an example of single cell sequence linkage by ligase chainreaction combined with overlap extension polymerase chain reaction, asapplied to a method for noninvasive prenatal diagnosis, according to oneembodiment of the invention.

FIG. 27 shows an example of hybridization of primers and target nucleicacids in a single cell sequence linkage by ligase chain reactioncombined with overlap extension polymerase chain reaction, as applied toa method for noninvasive prenatal diagnosis, according to one embodimentof the invention. The process is carried out in an emulsion droplet orreaction container (k).

FIG. 28 shows an example of resulting amplicons produced in a singlecell sequence linkage by ligase chain reaction combined with overlapextension polymerase chain reaction, as applied to a method fornoninvasive prenatal diagnosis, according to one embodiment of theinvention.

FIG. 29 shows hybridization of overlapping complementary regions of theresulting amplicons, and overlap extension polymerase chain reaction, asapplied to a method for noninvasive prenatal diagnosis, according to oneembodiment of the invention.

FIG. 30 shows the resulting amplicons from the overlap extensionpolymerase chain reaction, as applied to a method for noninvasiveprenatal diagnosis, according to one embodiment of the invention. Theend product is a library of “major amplicons”, or linked loci, which canthen be sequenced in bulk.

FIG. 31 shows a simplified workflow for high-throughput generation ofTCRβ repertoire libraries, according to one embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Briefly, and as described in more detail below, described herein aremethods and systems for massively parallel genetic analysis of singlecells in emulsion droplets or reaction containers. Genetic loci ofinterest are targeted in a single cell using specially-designed probes,and a fusion complex is formed by molecular linkage and amplificationtechniques. Multiple genetic loci can be targeted, and many sets ofprobes can be multiplexed by PCR into a single analysis, such thatseveral loci or even the entire transcriptome or genome is analyzed.

The invention is useful for analyzing genetic information in singlecells in a high-throughput, parallel fashion for a large quantity ofcells (10⁴ or greater cells). The invention is also useful for tracinggenetic information back to a cell or population of cells using uniquebarcode sequences.

DEFINITIONS

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

The term “cell” refers to a functional basic unit of living organisms. Acell includes any kind of cell (prokaryotic or eukaryotic) from a livingorganism. Examples include, but are not limited to, mammalianmononuclear blood cells, yeast cells, or bacterial cells.

The term “polymerase chain reaction” or PCR refers to a molecularbiology technique for amplifying a DNA sequence from a single copy toseveral orders of magnitude (thousands to millions of copies). PCRrelies on thermal cycling, which requires cycles of repeated heating andcooling of the reaction for DNA melting and enzymatic replication of theDNA. Primers (short DNA fragments) containing sequences complementary tothe target region of the DNA sequence and a DNA polymerase are keycomponents to enable selective and repeated amplification. As PCRprogresses, the DNA generated is itself used as a template forreplication, setting in motion a chain reaction in which the DNAtemplate is exponentially amplified. A heat-stable DNA polymerase, suchas Taq polymerase, is used. The thermal cycling steps are necessaryfirst to physically separate the two strands in a DNA double helix at ahigh temperature in a process called DNA melting. At a lowertemperature, each strand is then used as the template in DNA synthesisby the DNA polymerase to selectively amplify the target DNA. Theselectivity of PCR results from the use of primers that arecomplementary to the DNA region targeted for amplification underspecific thermal cycling conditions.

The term “reverse transcriptase polymerase chain reaction” or RT-PCRrefers to a type of PCR reaction used to generate multiple copies of aDNA sequence. In RT-PCR, an RNA strand is first reverse transcribed intoits DNA complement (complementary DNA or cDNA) using the enzyme reversetranscriptase, and the resulting cDNA is amplified using traditional PCRtechniques.

The term “ligase chain reaction” or LCR refers to a type of DNAamplification where two DNA probes are ligated by a DNA ligase, and aDNA polymerase is used to amplify the resulting ligation product.Traditional PCR methods are used to amplify the ligated DNA sequence.LCR provides greater specificity compared with PCR.

The term “emulsion droplet” or “emulsion microdroplet” refers to adroplet that is formed when two immiscible fluids are combined. Forexample, an aqueous droplet can be formed when an aqueous fluid is mixedwith a non-aqueous fluid. In another example, a non-aqueous fluid can beadded to an aqueous fluid to form a droplet. Droplets can be formed byvarious methods, including methods performed by microfluidics devices orother methods, such as injecting one fluid into another fluid, pushingor pulling liquids through an orifice or opening, forming droplets byshear force, etc. The droplets of an emulsion may have any uniform ornon-uniform distribution. Any of the emulsions disclosed herein may bemonodisperse (composed of droplets of at least generally uniform size),or may be polydisperse (composed of droplets of various sizes). Ifmonodisperse, the droplets of the emulsion may vary in volume by astandard deviation that is less than about plus or minus 100%, 50%, 20%,10%, 5%, 2%, or 1% of the average droplet volume. Droplets generatedfrom an orifice may be monodisperse or polydisperse. An emulsion mayhave any suitable composition. The emulsion may be characterized by thepredominant liquid compound or type of liquid compound that is used. Thepredominant liquid compounds in the emulsion may be water and oil. “Oil”is any liquid compound or mixture of liquid compounds that is immisciblewith water and that has a high content of carbon. In some examples, oilalso may have a high content of hydrogen, fluorine, silicon, oxygen, orany combination thereof, among others. For example, any of the emulsionsdisclosed herein may be a water-in-oil (W/O) emulsion (i.e., aqueousdroplets in a continuous oil phase). The oil may be or include at leastone silicone oil, mineral oil, fluorocarbon oil, vegetable oil, or acombination thereof, among others. Any other suitable components may bepresent in any of the emulsion phases, such as at least one surfactant,reagent, sample (i.e., partitions thereof), buffer, salt, ionic element,other additive, label, particles, or any combination thereof.

“Droplet” refers to a small volume of liquid, typically with a sphericalshape or as a slug that fills the diameter of a microchannel,encapsulated by an immiscible fluid. The volume of a droplet, and/or theaverage volume of droplets in an emulsion, may be less than about onemicroliter (i.e., a “microdroplet”) (or between about one microliter andone nanoliter or between about one microliter and one picoliter), lessthan about one nanoliter (or between about one nanoliter and onepicoliter), or less than about one picoliter (or between about onepicoliter and one femtoliter), among others. A droplet may have adiameter (or an average diameter) of less than about 1000, 100, or 10micrometers, or of about 1000 to 10 micrometers, among others. A dropletmay be spherical or nonspherical. In some embodiments, the droplet has avolume and diameter that is large enough to encapsulate a cell.

The term “barcode” refers to a nucleic acid sequence that is used toidentify a single cell or a subpopulation of cells. In some embodiments,a barcode sequence is used to identify a particular organism or aspecies. As described below, barcode sequences can be introduced into acell, linked by various amplification methods to a target nucleic acidof interest, and used to trace back the amplicon to the cell. Barcodesequences can be flanked by universal sequences that can be used toamplify libraries of barcodes using universal primer pairs. The barcodesequences can be contained within a circular or linear double-strandedmolecule, or in a single-stranded linear molecule.

The term “bulk sequencing” or “next generation sequencing” or “massivelyparallel sequencing” refers to any high throughput sequencing technologythat parallelizes the DNA sequencing process. For example, bulksequencing methods are typically capable of producing more than onemillion polynucleic acid amplicons in a single assay. The terms “bulksequencing,” “massively parallel sequencing,” and “next generationsequencing” refer only to general methods, not necessarily to theacquisition of greater than 1 million sequence tags in a single run. Anybulk sequencing method can be implemented in the invention, such asreversible terminator chemistry (e.g., Illumina), pyrosequencing usingpolony emulsion droplets (e.g., Roche), ion semiconductor sequencing(IonTorrent), single molecule sequencing (e.g., Pacific Biosciences),massively parallel signature sequencing, etc.

The term “in situ” refers to examining a biological phenomenon in theenvironment in which it occurs e.g., the practice of in situhybridization refers to hybridization of a probe to a nucleic acidtarget with the cell still intact.

The term “in vivo” refers to processes that occur in a living organism.

The term “mammal” as used herein includes both humans and non-humans andinclude, but is not limited to, humans, non-human primates, canines,felines, murines, bovines, equines, and porcines.

The term “T cell” refers to a type of cell that plays a central role incell-mediated immune response. T cells belong to a group of white bloodcells known as lymphocytes and can be distinguished from otherlymphocytes, such as B cells and natural killer T (NKT) cells by thepresence of a T cell receptor (TCR) on the cell surface. T cellsresponses are antigen-specific and are activated by foreign antigens. Tcells are activated to proliferate and differentiate into effector cellswhen the foreign antigen is displayed on the surface of theantigen-presenting cells in peripheral lymphoid organs. T cellsrecognize fragments of protein antigens that have been partly degradedinside the antigen-presenting cell. There are two main classes of Tcells-cytotoxic T cells and helper T cells. Effector cytotoxic T cellsdirectly kill cells that are infected with a virus or some otherintracellular pathogen. Effector helper T cells help to stimulate theresponses of other cells, mainly macrophages, B cells and cytotoxic Tcells.

The term “B cell” refers to a type of lymphocyte that plays a large rolein the humoral immune response (as opposed to the cell-mediated immuneresponse, which is governed by T cells). The principal functions of Bcells are to make antibodies against antigens, perform the role ofantigen-presenting cells (APCs) and eventually develop into memory Bcells after activation by antigen interaction. B cells are an essentialcomponent of the adaptive immune system.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

Methods of the Invention

I. Methods of Massively Parallel Single Cell Molecular Analysis

A. Microfluidics Methods for Generating Single Cell Emulsion Droplets

In some embodiments, a microfluidic device is used to generate singlecell emulsion droplets. The microfluidic device ejects single cells inaqueous reaction buffer into a hydrophobic oil mixture. The device cancreate thousands of emulsion microdroplets per minute. After theemulsion microdroplets are created, the device ejects the emulsionmixture into a trough. The mixture can be pipetted or collected into astandard reaction tube for thermocycling.

Custom microfluidics devices for single-cell analysis are routinelymanufactured in academic and commercial laboratories (Kintses et al.,2010 Current Opinion in Chemical Biology 14:548-555). For example, chipsmay be fabricated from polydimethylsiloxane (PDMS), plastic, glass, orquartz. In some embodiments, fluid moves through the chips through theaction of a pressure or syringe pump. Single cells can even bemanipulated on programmable microfluidic chips using a customdielectrophoresis device (Hunt et al., 2008 Lab Chip 8:81-87). In oneembodiment, a pressure-based PDMS chip comprised of flow-focusinggeometry manufactured with soft lithographic technology is used(Dolomite Microfluidics (Royston, UK)) (Anna et al., 2003 AppliedPhysics Letters 82:364-366). The stock design can typically generate10,000 aqueous-in-oil microdroplets per second at size ranges from10-150 μm in diameter. In some embodiments, the hydrophobic phase willconsist of fluorinated oil containing an ammonium salt ofcarboxy-perfluoropolyether, which ensures optimal conditions formolecular biology and decreases the probability of droplet coalescence(Johnston et al., 1996 Science 271:624-626). To measure periodicity ofcell and droplet flow, images are recorded at 50,000 frames per secondusing standard techniques, such as a Phantom V7 camera or Fastec InLine(Abate et al., 2009 Lab Chip 9:2628-31).

The microfluidic system can optimize microdroplet size, input celldensity, chip design, and cell loading parameters such that greater than98% of droplets contain a single cell. There are three common methodsfor achieving such statistics: (i) extreme dilution of the cellsolution; (ii) fluorescent selection of droplets containing singlecells; and (iii) optimization of cell input periodicity. For eachmethod, the metrics for success include: (i) encapsulation rate (i.e.,the number of drops containing exactly one cell); (ii) the yield (i.e.,the fraction of the original cell population ending up in a dropcontaining exactly one cell); (iii) the multi-hit rate (i.e., thefraction of drops containing more than one cell); (iv) the negative rate(i.e., the fraction of drops containing no cells); and (v) encapsulationrate per second (i.e., the number of droplets containing single cellsformed per second).

In some embodiments, single cell emulsions are generated by extreme celldilution. Under disordered conditions, the probability that amicrodroplet will contain k cells is given by the Poisson distribution:

${{f\left( {k;\lambda} \right)} = \frac{\lambda^{k}^{- \lambda}}{k!}},$

-   -   where e is the natural logarithm and the expected number of        occurrences in the interval is λ. Thus, for P(k=1)≈0.98, the        cell solution must be extremely dilute, such that λ≈0.04 and        only 3.84% of all drops contain a single cell.

In some embodiments, a simple microfluidic chip with a drop-makingjunction is used, such that an aqueous stream flows through a 10 μmsquare nozzle and dispenses the aqueous-in-oil emulsion mixtures into areservoir. The emulsion mixture can then be pipetted from the reservoirand thermocycled in standard reaction tubes. This method will producepredictably high encapsulation rates and low multi-hit rates, but a lowencapsulation rate per second. A design that can achieve filled dropletthroughput of 1000 Hz is capable of sorting up to 10⁶ cells in less than17 minutes.

Fluorescence techniques can also be used to sort microdroplets withparticular emission characteristics (Baroud et al., 2007 Lab Chip7:1029-1033; Kintses et al., 2010 Current Opinion in Chemical Biology14:548-555). In these studies, chemical methods are used to stain cells.In some embodiments, autofluorescence is used to select microemulsionsthat contain cells. A fluorescent detector reduces the negative rateresulting from extreme cell dilution. A microfluidic device can also beequipped with a laser directed at a “Y” sorting junction downstream ofthe cell encapsulation junction. The Y junction has a “keep” and a“waste” channel. A photomultiplier tube is used to collect thefluorescence of each drop as it passes the laser. The voltage differenceis calibrated between empty drops and drops with at least one cell.Next, when the device detects a droplet that contains at least one cell,and electrodes at the Y sorting junction create a field gradient bydielectrophoresis (Hunt et al., 2008 Lab on a Chip 8:81-87) and pushdroplets containing cells in to the keep channel. The microfluidicdevice uses extreme cell dilution to control the multi-hit rate andfluorescent cell sorting to reduce the negative rate.

In some embodiments, input cell flow is aligned with droplet formationperiodicity, such that greater than 98% of droplets contain a singlecell (Edd et al., 2008 Lab Chip 8:1262-1264; Abate et al., 2009 Lab Chip9:2628-31). In these microfluidic devices, a high-density suspension ofcells is forced through a high aspect-ratio channel, such that the celldiameter is a large fraction of the channel's width. The chip isdesigned with a 27 μm×52 μm rectangular microchannel that flows cellsinto microdroplets at >10 μL/min (Edd et al., 2008 Lab Chip8:1262-1264). A number of input channel widths and flow rates are testedto arrive at an optimal solution.

In some embodiments, cells with different morphology might behavedifferently in the microchannel stream of the microfluidic device,confounding optimization of the technique when applied to clinicalbiological samples. To address this issue, a field gradientperpendicular to the microchannel by dielectrophoresis is induced.Dielectrophoresis pulls the cells to one side of the microchannel,creating in-channel ordering that is independent of cell morphology.This method requires substantial optimization of charge and flow rateand a more complicated chip and device design, so this method may benecessary if existing methodologies fail to perform for certain celltypes.

The emulsion microdroplet mixtures are pipetted from the trough in themicrofluidic device to a reaction tube for thermocycling. Afterthermocycling the emulsions, a number of methods might achieve emulsionreversal to recover the aqueous phase of the reaction. Twostraightforward reversal processes that have been used by priorinvestigators are flash-freezing in liquid nitrogen for 10 seconds(Kliss et al., 2008 Analytical Chem 80:8975-8981) and passage through a15 μm mesh filter (Zeng et al., 2010 Analytical Chem 82: 3183-90).Emulsion reversal can also be achieved using commercially availablereagents designed for this purpose (Brouzes et al., 2009 PNAS106:14195-200). Success of the emulsion reversal is assessed byvisualization of the aqueous and hydrophobic phases under a microscope.

In some embodiments, the methods of the invention use single cells inreaction containers, rather than emulsion droplets. Examples of suchreaction containers include 96 well plates, 0.2 mL tubes, 0.5 mL tubes,1.5 mL tubes, 384-well plates, 1536-well plates, etc.

B. Methods for Molecular Linkage in Single Cell Emulsions and MassivelyParallel Sequencing

1) Molecular Linkage Using Polymerase Chain Reaction (PCR)

PCR is used to amplify many kinds of sequences, including but notlimited to SNPs, short tandem repeats (STRs), variable protein domains,methylated regions, and intergenic regions. Methods for overlapextension PCR are used to create fusion amplicon products of severalindependent genomic loci in a single tube reaction (Johnson et al., 2005Genome Research 15:1315-24; U.S. Pat. No. 7,749,697).

In some embodiments, at least two nucleic acid target sequences (e.g.,first and second nucleic acid target sequences, or first and secondloci) are chosen in the cell and designated as target loci. Forward andbackward primers are designed for each of the two nucleic acid targetsequences, and the primers are used to amplify the target sequences.“Minor” amplicons are generated by amplifying the two nucleic acidtarget sequences separately, and then fused by amplification to create afusion amplicon, also known as a “major” amplicon. In one embodiment, a“minor” amplicon is a nucleic acid sequence amplified from a targetgenomic loci, and a “major” amplicon is a fusion complex generated fromsequences amplified between multiple genomic loci. Exemplary primersthat can be used for generating minor and major amplicons are listed inTable 2. These primers are used for multiplexed amplification of asingle cell's TCRβ and then linkage of the TCRβ to immune effectortargets IL-2, IL-4, INFG, TBX21, FOXP3, or TNFA. In one embodiment, SEQID NOs: 1-57 are pooled together with primers for a single immuneeffector target, e.g., SEQ ID NOs: 68 and 69.

The method uses “inner” primers (i.e., the reverse primer for the firstlocus and the forward primer for the second locus) comprising of onedomain that hybridizes with a minor amplicon and a second domain thathybridizes with a second minor amplicon. “Inner” primers are a limitingreagent, such that during the exponential phase of PCR, inner primersare exhausted, driving overlapping domains in the minor amplicons toanneal and create major amplicons.

PCR primers are designed against targets of interest using standardparameters, i.e., melting temperature (Tm) of approximately 55-65° C.,and with a length 20-50 nucleotides. The primers are used with standardPCR conditions, for example, 1 mM Tris-HCl pH 8.3, 5 mM potassiumchloride, 0.15 mM magnesium chloride, 0.2-2 μM primers, 200 μM dNTPs,and a thermostable DNA polymerase. Many commercial kits are available toperform PCR, such as Platinum Taq (Life Technologies), Amplitaq Gold(Life Technologies), Titanium Taq (Clontech), Phusion polymerase(Finnzymes), HotStartTaq Plus (Qiagen). Any standard thermostable DNApolymerase can be used for this step, such as Taq polymerase or theStoffel fragment.

In one embodiment, a set of nucleic acid probes (or primers) are used toamplify a first target nucleic acid sequence and a second target nucleicacid sequence to form a fusion complex. The first probe includes asequence that is complementary to a first target nucleic acid sequence(e.g., the 5′ end of the first target nucleic acid sequence). The secondprobe includes a sequence that is complementary to the first targetnucleic acid sequence (e.g., the 3′ end of the first target nucleic acidsequence) and a second sequence that is complementary to an exogenoussequence. In some embodiments, the exogenous sequence is a non-humannucleic acid sequence and is not complementary to either of the targetnucleic acid sequences. The first and second probes are the forwardprimer and reverse primer for the first target nucleic acid sequence.

The third probe includes a sequence that is complementary to the portionof the second probe that is complementary to the exogenous sequence anda sequence that is complementary to the second target nucleic acidsequence (e.g., the 5′ end of the second target nucleic acid sequence).The fourth probe includes a sequence that is complementary to the secondtarget nucleic acid sequence (e.g., the 3′ end of the second targetnucleic acid sequence). The third probe and the fourth probe are theforward and reverse primers for the second target nucleic acid sequence.

The second and third probes are also called the “inner” primers of thereaction (i.e., the reverse primer for the first locus and the forwardprimer for the second locus) and are limiting in concentration, (e.g.,0.01 μM for the inner primers and 0.1 μM for all other primers). Thiswill drive amplification of the major amplicon preferentially over theminor amplicons. The first and fourth probes are called the “outer”primers.

The first and second nucleic acid sequences are amplified independently,such that the first nucleic acid sequence is amplified using the firstprobe and the second probe, and the second nucleic acid sequence isamplified using the third probe and the fourth probe. Next, a fusioncomplex is generated by hybridizing the complementary sequence regionsof the amplified first and second nucleic acid sequences and amplifyingthe hybridized sequences using the first and fourth probes. This iscalled overlap extension PCR amplification.

During overlap extension PCR amplification, the complementary sequenceregions of the amplified first and second nucleic acid sequences act asprimers for extension on both strands and in each direction by DNApolymerase molecules. In subsequent PCR cycles, the outer primers primethe full fused sequence such that the fused complex is duplicated by DNApolymerase. This method produces a plurality of fusion complexes.

FIGS. 5-6 show an example of the single cell sequence linkage byintracellular overlap extension polymerase chain reaction, according toone embodiment of the invention. In FIG. 5A, a forward primer (a)targets one locus of a first target nucleic acid (g). A reverse primer(b) targets another locus of the first target nucleic acid (g) and has aregion of complementarity (c) to a region (d) of the forward primer (e).The forward primer (e) has a region of complementarity to the secondtarget nucleic acid (h) and the reverse primer (f) targets anotherregion of the second target nucleic acid (h). FIG. 5B illustrates anexample of the hybridization of the probes (a, b, e and f) to respectivetarget nucleic acids (g and h), according to one embodiment of theinvention. FIG. 6A illustrates an example of the complementary regions(c) and (d) between amplicons (g) and (h), according to one embodimentof the invention. FIG. 6B shows linkage amplification of the amplicons(g) and (h) using polymerase (e) to create a linked major amplicon (i).The end product is a library of “major amplicons” that include thelinked amplicons (g) and (h), which can be sequenced in bulk. The stepsof FIGS. 5-6 can be performed in a reaction container or an emulsiondroplet.

In some embodiments, multiple loci are targeted in a single cell, andmany sets of probes can be multiplexed into a single analysis, such thatseveral loci or even the entire transcriptome or genome is analyzed.Multiplex PCR is a modification of PCR that uses multiple primer setswithin a single PCR mixture to produce amplicons of varying sizes thatare specific to different DNA sequences. By targeting multiple genes atonce, additional information may be gained from a single test run thatotherwise would require several times the reagents and more time toperform. In one embodiment, 10-20 different transcripts are targeted ina single cell and linked to a second target nucleic acid (e.g., linkedto a variable region such as a mutated gene sequence, a barcode, or animmune variable region).

In one embodiment, single cells are encapsulated in aqueous-in-oilpicoliter microdroplets. The droplets enable compartmentalization ofreactions such that molecular biology can be performed on millions ofsingle cells in parallel. Monodisperse aqueous-in-oil microdroplets canbe generated on microfluidic devices at size ranges from 10-150 μm indiameter. Alternatively, droplets can be generated by vortexing or by aTissueLyser (Qiagen). Two embodiments of oil and aqueous solutions forcreating PCR microdroplets are: (i) PCR buffer that contains 0.5 μg/μLbovine serum albumin (New England Biolabs) combined with mixture offluorocarbon oil (3M), Krytox 157FSH surfactant (Dupont), and PicoSurf(Sphere Microfluidics); and (ii) PCR buffer with 0.1% Tween 20 (Sigma)combined with a mixture of light mineral oil (Sigma), EM90 (Evonik), andTriton X-100 (Sigma). Several replicate assays quantifying 1 millionamplicons by next-generation sequencing have shown that both chemistriesform monodisperse microdroplets that are >99.98% stable after 40 cyclesof PCR. PCR can occur in a standard thermocycling tube, a 96-well plate,or a 384-well plate, using a standard thermocycler (Life Technologies).PCR can also occur in heated microfluidic chips, or any other kind ofcontainer that can hold the emulsion and transfer heat.

After thermocycling and PCR, the amplified material must be recoveredfrom the emulsion. In one embodiment, ether is used to break theemulsion, and then the ether is evaporated from the aqueous/ether layerto recover the amplified DNA in solution. Other methods include adding asurfactant to the emulsion, flash-freezing with liquid nitrogen, andcentrifugation.

Once the linked and amplified products are recovered from the emulsion,there are a number of methods to prepare the product for bulksequencing. In one embodiment, the major amplicon is isolated from theminor amplicons using gel electrophoresis. If yield is not sufficient,the major amplicon is amplified again using PCR and the two outerprimers. This material can then be sequenced directly using bulksequencing. In some embodiments, the outer primers are used to producemolecules than can be sequenced directly. In other embodiments, adaptersmust be added to the major amplicon before bulk sequencing. Once thesequencing library is synthesized, bulk sequencing can be performedusing standard methods and without significant modification.

2) Molecular Linkage Using Reverse Transcriptase Polymerase ChainReaction (RT-PCR)

The overlap extension PCR method adapts to single tube overlap extensionRT-PCR, which amplifies DNA from RNA transcripts. The RT-PCR methodcombines cDNA synthesis and PCR in enclosed tubes without bufferexchange or reagent addition between the molecular steps. Thermostablereverse transcriptase (RT) enzymes are used that withstand temperaturesgreater than 95° C., though thermostable RT is not necessary if firststrand cDNA synthesis occurs prior to PCR amplification. For example,both ThermoScript RT (Lucigen) and GeneAmp Thermostable rTth (LifeTechnologies) are designed and used in single-tube reverse transcriptasePCR. In one embodiment, a set of nucleic acid probes (or primers) areused to amplify a first target nucleic acid sequence and a second targetnucleic acid sequence to form a fusion complex. The first target nucleicacid sequence or the second target nucleic acid sequence is RNA.

The first probe includes a sequence that is complementary to a firsttarget nucleic acid sequence (e.g., the 5′ end of the first targetnucleic acid sequence). The second probe includes a sequence that iscomplementary to the first target nucleic acid sequence (e.g., the 3′end of the first target nucleic acid sequence) and a second sequencethat is complementary to an exogenous sequence. In some embodiments, theexogenous sequence is a non-human nucleic acid sequence and is notcomplementary to either of the target nucleic acid sequences. The firstand second probes are the forward primer and reverse primer for thefirst target nucleic acid sequence.

The third probe includes a sequence that is complementary to the portionof the second probe that is complementary to the exogenous sequence anda sequence that is complementary to the second target nucleic acidsequence (e.g., the 5′ end of the second target nucleic acid sequence).The fourth probe includes a sequence that is complementary to the secondtarget nucleic acid sequence (e.g., the 3′ end of the second targetnucleic acid sequence). The third probe and the fourth probe are theforward and reverse primers for the second target nucleic acid sequence.

The second and third probes are also called the “inner” primers of thereaction (i.e., the reverse primer for the first locus and the forwardprimer for the second locus) and are limiting in concentration, (e.g.,0.01 μM for the inner primers and 0.1 μM for all other primers). Thiswill drive amplification of the major amplicon preferentially over theminor amplicons. The first and fourth probes are called the “outer”primers.

The method includes amplifying using RT-PCR the first and second nucleicacid sequences independently, such that the first nucleic acid sequenceis amplified using the first probe and the second probe, and the secondnucleic acid sequence is amplified using the third probe and the fourthprobe. Using overlap extension PCR amplification, a fusion complex isgenerated by hybridizing the complementary sequence regions of theamplified first and second nucleic acid sequences and amplifying thehybridized sequences using the first and fourth probes. (See FIGS. 5-6).

3) Molecular Linkage Using Ligase Chain Reaction

Ligase chain reaction (LCR) is used to target and amplify genetic lociof interest (Landegren et al., 1988 Science 241:1077-1080; Benjamin etal., 2003 Methods in Molecular Biology 226: 135-149; U.S. Pat. No.6,235,472). In ligase chain reaction, two polynucleic acid probes targeta polynucleic acid locus of interest. Upon hybridization, the two probesare ligated by a ligase enzyme. In contrast with PCR, LCR amplifies bothRNA and DNA, facilitating many different kinds of multiplexed analysis.Another notable advantage of ligase chain reaction is the capacity forallele-specific amplification. Whereas PCR amplifies both alleles for aparticular variant, the ligation process of LCR is allele-specific.

In some embodiments, LCR probes are used as a molecular “switch.” Forexample, if millions of single cells are screened for a particularvariant, only cells that include that variant will produce majoramplicons. LCR is used to perform genetic analysis only on cells thatcontain a particular sequence of interest. Cells that lack the sequenceof interest are not substantially amplified and are therefore silent inthe reaction. LCR can also be multiplexed more efficiently than PCR,using hundreds of probes targeting hundreds of genetic loci in a singlecell microdroplet or intracellular reaction.

In one embodiment, a single tube-single buffer overlap extension LCR/PCRreaction mixture is formulated using DNA and/or RNA, LCR probes, the PCRprimers, Ampligase (Epicentre), a DNA polymerase such as Stoffelfragment (Life Technologies), and reaction buffer (20 mM Tris-HCl, 25 mMKCl, 10 mM MgCl₂, 0.5 mM NAD, 0.01% Triton X-100). The method combinesLCR with overlap extension PCR to leverage the benefits of both LCR andPCR (FIGS. 7-9). The “inner” probes are added at 1/10 of theconcentration of the other oligonucleotides in the reaction such thatthey become a limiting reagent at later cycles. For the initialannealing and ligation, the mixtures can be incubated for 4 minutes at20° C., 5 minutes at 95° C., and 15 minutes at 60° C. Standard PCRthermocycling conditions are used to amplify the minor and majoramplicons (95° C., 5 minutes; [95° C., 30 seconds; 60° C., 30 seconds;72° C., 30 seconds]×30 cycles). The major amplicon is amplified furtherby gel size selection and another round of amplification using the outerprimers only. FIGS. 7A and 7B illustrate an example of single cellsequence linkage by intracellular ligase chain reaction combined withoverlap extension polymerase chain reaction, according to one embodimentof the invention. A forward LCR primer (a) targets one locus of a firsttarget nucleic acid (g). A reverse LCR primer (b) targets another locusof the first target nucleic acid (g) and has a region of complementarity(c) to a region (d) of the forward primer (e). The forward LCR primer(e) has a region of complementarity to the second target nucleic acid(h) and the reverse LCR primer (f) targets another region of the secondtarget nucleic acid (h).

FIG. 8A shows another example of the complementary regions betweenamplicons (a) and (d), according to one embodiment of the invention.FIG. 8B shows linkage amplification of the amplicons using polymerase(e) to create a linked major amplicon. The steps of FIGS. 7 and 8 can beperformed in a reaction container or an emulsion droplet.

FIG. 9A shows an example of a linked amplicon (f), according to oneembodiment of the invention. FIG. 9B shows the resulting ampliconproduced from the steps shown in FIGS. 8A and 8B. The end product can bea library of “major amplicons” and are sequenced in bulk.

In another embodiment, the single cell sequence linkage by intracellularligase chain reaction combined with overlap extension polymerase chainreaction is performed with the following set of probes: a first LCRprobe comprising a sequence that is complementary to a first targetnucleic acid subsequence, a second probe comprising a sequence that iscomplementary to a second subsequence of the first target nucleic acidand a second sequence that is complementary to an exogenous sequence, athird probe comprising the exogenous sequence and a sequence that iscomplementary to a first subsequence of a second target nucleic acid,and a fourth probe comprising a sequence that is complementary to asecond subsequence of the second target nucleic acid sequence. Themethod includes isolating the single cells with at least one set ofnucleic acid probes. The first and second probes are hybridized to thefirst nucleic acid and ligated by a ligase enzyme. Similarly, the thirdand fourth probes are hybridized to the second target nucleic acid andligated by a ligase enzyme. Then, the ligated probes for the first andsecond target nucleic acids are hybridized across the complementaryregion comprising the exogenous sequence and overlap extension PCR isused to generating a fused complex. The fused complexes can be bulksequenced.

4) Molecular Linkage Using Padlock Probes

A padlock probe is a circularized, single stranded DNA or RNA moleculewith complementarity to a sequence target of interest (Hardenbol et al.,2003 Nature Biotechnology 21:673-678; U.S. Pat. No. 6,858,412). Afterhybridization to the target molecules, a polymerase fills the gapbetween the two ends of the probe, and a ligase completes thepolynucleotide chain to form a circularized polynucleotide molecule. Thecircularized molecule can then be amplified with multiple displacementamplification (MDA). MDA is an isothermal amplification method thatfunctions by annealing single stranded polynucleotides to the template,followed by DNA synthesis by a high fidelity enzyme such as φ-29polymerase. Inverse PCR can also be used to amplify only thecircularized molecules because PCR primers that amplify the circularizedmolecules will not amplify the single stranded probes (U.S. Pat. No.6,858,412).

A notable advantage of padlock probes over PCR is the capacity forallele-specific amplification. Whereas PCR amplifies both alleles for aparticular variant, the ligation process of padlock probes isallele-specific. As with LCR, padlock probes are used as a molecular“switch.” If millions of single cells are screened for a particularvariant, only cells that include that variant will produce majoramplicons. Thus, padlock probes are used to perform genetic analysisonly on cells that contain a particular sequence of interest. Also, incertain embodiments, padlock probes are highly multiplexed, with tens ofthousands of probe types targeting tens of thousands of genetic loci ina single cell microdroplet or intracellular reaction (See U.S. Pat. No.6,858,412).

Padlock probes are typically hybridized to targets by cycling at least20 times between 95° C. for 5 min and 55° C. for 20 min (Baner et al.,2003 Nucleic Acids Research 31: e103). The single nucleotide gaps arethen filled with Stoffel polymerase and ligase, such as Tth ligase orAmpligase (Epicentre). The circularized probes are then be amplifiedusing PCR with universal primers. When multiplexed for overlap extensionPCR, two sets of universal primers are used, one for each padlock probetype. The universal primers contain sequence regions of overlap, whichenables standard overlap extension PCR following initial sequencecapture by the padlock probes. (See FIGS. 10-14). The probes can also beengineered to contain the appropriate primer sequences for bulksequencing, so the library is sequenced directly after PCRamplification.

FIG. 10 illustrates an example of the components required for a singlecell sequence linkage by padlock probes combined with overlap extensionpolymerase chain reaction, according to one embodiment of the invention.FIG. 11 shows the complementary regions between a first padlock probe(a) and the first target nucleic acid (c) and between a second padlockprobe (b) and a second target nucleic acid (d) in a single cell,according to one embodiment of the invention. The reaction componentscan be contained in a physical reaction container or an emulsion droplet(k). The first padlock probe (a) includes two separate regions that arecomplementary to the first target nucleic acid (c). The second padlockprobe (b) includes two separate regions that are complementary to asecond target nucleic acid (d). A polymerase and a ligase are used (m)to amplify and ligate the gap between complementary regions of thepadlock probes (a) and (b).

FIG. 12 illustrates the resulting circularized amplicons (g) and (h) andthe primers that are used to amplify the circularized amplicons,according to one embodiment of the invention. A forward primer (a) and areverse primer (i) are used to amplify circular amplicon (g). Forwardand reverse primers (j) and (f) are used to amplify circular amplicon(h). Primer (i) has a region (b) that is complementary to a region ofamplicon (g) and a region (c) that is complementary to region (d) ofprimer (j). Primer (j) has a region (e) that is complementary to theamplicon (h) and a region (d) that is complementary to region (c) ofprimer (i).

FIG. 13 is an example of the resulting amplicons from amplification ofthe circular probes (g) and (h), according to one embodiment of theinvention. In this figure, region (a) is complementary to amplicon (g)and region (b) is complementary to region (c). Region (d) iscomplementary to amplicon (h) and region (c) is complementary to region(b).

FIG. 14 is an example of overlap extension PCR amplification of theamplicons using a polymerase (e), according to one embodiment of theinvention. The resulting amplicon (f) includes sequences (a), (d), andthe overlapping sequences (b) and (c). The resulting amplicon (f) can beused for bulk sequencing. The steps can be performed in a reactioncontainer or an emulsion droplet (g).

5) Molecular Linkage Using Multiprobe Circularization

In some embodiments, multiprobe circularization can be used. Inmultiprobe circularization, two padlock probes target two genetic loci.After hybridization to the target molecules, a polymerase fills the gapbetween the ends of the two probes, and a ligase completes thepolynucleotide chains to form a circularized polynucleotide molecule.(See FIGS. 1A-1C). The circularized molecule can then be amplified withmultiple displacement amplification (MDA). Inverse PCR can also be usedto amplify only the circularized molecules, because PCR primers thatamplify the circularized molecules will not amplify the single strandedprobes (See FIGS. 2-3).

In one embodiment, the probes are hybridized to targets by cycling atleast 20 times between 95° C. for 5 min and 55° C. for 20 min (Baner etal., 2003 Nucleic Acids Research 31: e103). The single nucleotide gapsare filled with a Stoffel polymerase and ligase. The circularized probesare amplified using PCR with universal primers. When multiplexed foroverlap extension PCR, the two sets of universal primers are used, onefor each padlock probe type. The universal primers contain sequenceregions of overlap, which enables standard overlap extension PCRfollowing initial sequence capture by the padlock probes (FIGS. 2-3).The probes can also be engineered to contain the appropriate primersequences for bulk sequencing, so the library is sequenced directlyafter PCR amplification.

FIG. 1 shows an example of sequence linkage in a single cell byintra-cellular multiprobe circularization of a molecular complex,according to one embodiment of the invention. Each probe has a region ofcomplementarity to each of the target loci. The complex includes twonucleic acid probes (a and b) and two target nucleic acids (c and d).The single cell (e) can be contained in a reaction container or anemulsion droplet (j). FIG. 1A illustrates that the nucleic acid probe(a) has a first region (f) that is complementary to a region on thetarget nucleic acid (c), and a second region (g) that is complementaryto a region on the target nucleic acid (d). The nucleic acid probe (b)has a first region (h) that is complementary to a region on the targetnucleic acid (c) and a second region (i) that is complementary to aregion on the target nucleic acid (d). FIG. 1B illustrates an example ofsequence linkage in a single cell (also in a reaction container oremulsion droplet (j)) by intra-cellular multiprobe circularization of acomplex, according to one embodiment of the invention. The two nucleicacid probes (a and b) are hybridized to the complementary regions of thetwo target nucleic acids (c and d). FIG. 1C illustrates an example ofcircularization of a probe-target linkage complex occurs byamplification, according to one embodiment of the invention. In oneexample, a φ-29 polymerase mediated rolling circle amplification is usedto circularize the end regions (k) of the two nucleic acid probes (a)and (b).

FIG. 2 shows an example of amplification of a circularized probe-targetlinkage complex (a) using a polymerase (b), according to one embodimentof the invention. In some embodiments, a φ-29 polymerase is used in amediated rolling circle amplification, and copies (b and c) of thecircularized probe-target complex are generated. In addition, FIG. 3illustrates an example of amplification of a circularized probe-targetlinkage complex (a) using a polymerase (b) and primers (c and d),according to one embodiment of the invention. The primers (c and d) areused to amplify the region of the circularized probe-target complex thatis complementary to the target nucleic acid. Multiple copies (e) of alinear double-stranded polynucleic acid amplicon are generated andsequenced in bulk.

6) Methods Using Barcode Nucleic Acids

Bulk sequencing requires destruction of cells or emulsion microdroplets,such that all polynucleic acid analytes are pooled into a singlereaction mixture. Trace back of a particular sequence target from bulksequencing data to a particular cell is typically not possible. However,many applications will require trace back of sequences to their originalsingle cells. For example, an investigator may wish to analyze a cellpopulation for single cell expression patterns for two RNA transcripts.Overlap extension reverse transcriptase PCR amplification of two RNAtranscript targets followed by bulk sequencing is not adequate for suchan analysis because all of the transcripts are mixed together, andtranscripts from high-expressing cells are indistinguishable fromtranscripts from low-expressing cells. To address this problem,polynucleic acid barcodes are used. Each single cell emulsionmicrodroplet or physical reaction container contains a single uniqueclonal polynucleic acid barcode. This barcode is then linked to thetarget polynucleic acids (i.e., RNA transcripts), and is used to traceback the major amplicons to a single cell (See FIGS. 18-25). With traceback of each sequence to an original single cell, it is possible totabulate genetic data for each single cell, which then enables singlecell quantification (i.e., single cell gene expression levels).

In one embodiment, the linker barcode oligonucleotide is highly diluted,such that less than 1% of picoliter emulsion microdroplets carry morethan one linker barcode. This enables the linking of a single cell to asingle barcode. The linker barcode oligonucleotide is amplified by PCRusing universally primers inside each droplet, such that each dropletwill contain millions of copies of only one linker barcode sequence, andthat barcode will be unique to that droplet (FIGS. 18-21). The dilutionfollows Poisson statistics such that for P(k=1)≈0.99, the linkerbarcodes need to be diluted to λ≈0.01. The barcode is then physicallylinked to the target molecule by overlap extension PCR. Barcodes can beproduced by a number of methods. In one embodiment, a library of randomdecamers are subcloned into a plasmid vector (e.g., Life Technologies).This produces a mixed plasmid library with >1 million unique decamerbarcodes. Then, the plasmids are transformed into bacteria and 3,840clones are picked. The clones are sequenced by capillary sequencing(Sequetech) and archived in glycerol stocks on 384-well plates. Next,the clones are digested at restriction sites on either side of therandom decamer inserts to produce a ˜100 bp fragment. These fragmentsare then biotinylated using Klenow fragment with standard procedures.Washing between molecular steps is performed with the aid of Ampure beadtechnology (PerkinElmer). The biotinylated fragments are then be affixedto 17 μm diameter streptavidin beads (Life Technologies) in each well,producing 3,840 clonal populations of barcode beads. Nucleic acidamplification using bead emulsions is described in U.S. Pat. No.7,842,457.

In one embodiment, the method provides beads attached to barcode nucleicacid sequences. A library of random 15-mers is subcloned into a plasmidvector (Life Technologies). This produces a mixed plasmid librarywith >1 billion unique 15-mer barcodes. The biotinylated fragments arethen affixed to 17 μm diameter streptavidin beads (Life Technologies).The plasmid barcode mixture is diluted in PCR mix such that 99% of thedroplets that contain a plasmid will contain only a single clonalplasmid. The PCR mix contains biotinylated nucleotides, such thatamplified barcodes are biotinylated. Then, streptavidin beads are flowedinto this PCR mix to encapsulate single beads in microdroplets. At least10 million beads are typically encapsulated, and then the bead/plasmidmixes are thermocycled to amplify and biotinylate the barcodes. Thebarcoded beads are then recovered and can be used in the dropletbarcoding method.

In another embodiment, a microfluidic device injects beads coated withclonal linker barcode oligonucleotides into the single cell emulsionmicrodroplets. Such a device enables visualization of single beads andsingle cells in each drop, eliminating the requirement for highly dilutelinker barcode oligonucleotides. In this embodiment, PCR is also used toamplify the linker barcode oligonucleotide, such that each dropletcontains millions of copies of the same barcode sequence, but eachbarcode would be unique to a single microdroplet. The barcode is thenlinked to the target nucleic acid sequence using overlap extension PCR.During overlap extension PCR amplification, the complementary sequenceregions of the amplified first and second nucleic acid sequences act asprimers for extension on both strands in each direction by DNApolymerase molecules. In subsequent PCR cycles, the outer primers primethe full fused sequence such that it is duplicated by DNA polymerase.This method produces a plurality of fusion complexes.

In another embodiment, the method includes steps for providing a pool ofunique barcode sequences, where each barcode sequence is linked to aselection resistance gene, providing a population of single cells,transfecting the population of single cells with the pool of uniquebarcode sequences, selecting cells comprising a unique barcode sequenceand the selection resistance gene, and isolating each of the selectedcells into reaction containers or emulsion microdroplets. In someembodiments, the selection resistance gene encodes resistance togentamycin, neomycin, hygromycin, or puromycin. The selection resistancegene enables one to select cells that have incorporated the barcodesequence into the cell. Cells that lack the plasmid also lack theselection resistance gene and therefore are killed in the presence of amammalian selection chemical such as gentamycin, neomycin, hygromycin,or puromycin.

FIG. 15 illustrates an example of plasmid library deconvolution bybarcoded tailed end (5′-end barcoded) polymerase chain reaction, whichis followed by bulk sequencing and informatics, according to oneembodiment of the invention. The barcode sequence can be traced back toa well and plate position, the barcode sequence can then be traced to anucleic acid sequence, and the nucleic acid sequence is traced back to awell. Each of the primers in (a) and (b) have a 5′-end barcoded tag. Thetarget nucleic acids in (c) and (d) are amplified using the primers in(a) and (b). The steps can be performed in enclosed containers oremulsion droplets, as shown in (c) and (d). FIG. 16 also shows anexample of amplification (e, f) of two target nucleic acids (A and B)using primers that include barcode sequences, according to oneembodiment of the invention. The resulting amplicons that include thebarcode sequences are shown in (g) and (h). Moreover, FIG. 17illustrates a simplified example of tracing back a barcode sequence inan amplicon to a cell target (A or B), and tracing back the cell targetto a physical location (c, d) (e.g., a well), according to oneembodiment of the invention.

In addition, FIG. 18 illustrates the components for molecular linkagebetween two transcripts (g and h) and a molecular barcode sequence (k),according to one embodiment of the invention. The targets (g and h) canbe RNA transcripts, and the molecular barcode sequence (k) is flanked byuniversal priming sites. Only one copy of the molecular barcodeoligonucleotide is contained in the emulsion droplet or reactioncontainer (j), and universal PCR primers amplify the oligonucleotide toproduce a plurality of clonal barcode polynucleic acids. A forwardprimer (a) and reverse primer (m) are used to amplify target nucleicacid (g). A forward primer (n) and reverse primer (f) are used toamplify target nucleic acid (h). The reverse primer (m) includes aregion (b) that is complementary to the target nucleic acid (g) and aregion (c) that is complementary to region (d) on primer (n). Primer (n)includes a region (e) of complementarity to target nucleic acid (h) anda region (d) of complementarity to region (c) of primer (m). In someembodiments, more than two targets can be linked, and the targets canalso be DNA.

In addition, FIG. 19 shows an example of amplification of the targetnucleic acids (g and h) using primers as shown, according to oneembodiment of the invention. The forward primer (a) is complementary totarget nucleic acid (g), and the reverse primer (b) for the targetnucleic acid (g) includes a region (c) that is complementary to thebarcode sequence (k). Forward primer (e) and reverse primer (f) are usedto amplify target nucleic acid (h). The forward primer (e) includes aregion (d) that is complementary to the barcode sequence (k).

In FIG. 20, amplicons resulting after amplification of two targetnucleic acids and a barcode sequence (k) are shown, according to oneembodiment of the invention. FIG. 21 illustrates a fused amplicon thatincludes sequences of two target nucleic acids (g and h) and a barcodesequence (k) inside an emulsion droplet or reaction container (j),according to one embodiment of the invention. The fused (“major”)amplicon can be isolated by reverse emulsion and bulk sequenced. In FIG.22, the targets (g and h) can be RNA transcripts, and the molecularbarcode sequence (k) is flanked by universal priming sites. Only onecopy of the molecular barcode sequence (k) is contained in the singlecell emulsion droplet or reaction container (j), and universal PCRprimers amplify the oligonucleotide to produce a plurality of clonalbarcode polynucleic acids. Forward primer (a) and reverse primer (b) areused to amplify target nucleic acid (g). Forward primer (n) and reverseprimer (f) are used to amplify target nucleic acid (h). The reverseprimer (m) includes a region (b) that is complementary to the targetnucleic acid (g) and a region (c) that is complementary to region (d) onprimer (n). Primer (n) includes a region (e) of complementarity totarget nucleic acid (h)

and a region (d) of complementarity to region (c) of primer (m). In someembodiments, more than two targets can be linked, and the targets canalso be DNA.

FIG. 23 illustrates the forward and reverse primers that are used in amolecular linkage between two transcripts (g and h) and a molecularbarcode sequence (k) attached to a bead (m), according to one embodimentof the invention. Forward primer (a) and reverse primer (b) are used toamplify target nucleic acid (g). Forward primer (n) and reverse primer(f) are used to amplify target nucleic acid (h). The reverse primer (m)includes a region (b) that is complementary to the target nucleic acid(g) and a region (c) that is complementary to region (d) on primer (n).Primer (n) includes a region (e) of complementarity to target nucleicacid (h) and a region (d) of complementarity to region (c) of primer(m). The two target nucleic acids are complementary to a DNA sequence(l). FIG. 24 is an example of amplicons resulting after amplification oftwo target nucleic acids and a barcode sequence (k) attached to a bead(m), according to one embodiment of the invention. FIG. 25 illustrates afused amplicon that includes sequences of two target nucleic acids (gand h) and a barcode sequence (k), inside an emulsion droplet orreaction container (j), according to one embodiment of the invention.The fused (“major”) amplicon can be isolated by reverse emulsion andbulk sequenced. FIGS. 24-25 illustrate an example of amplicons resultingafter amplification of two target nucleic acids and a barcode sequence(k) attached to a bead (m), according to one embodiment of theinvention.

7) Methods Using Combination Amplification

Targeting and amplification of genetic loci in cells can be performedusing PCR, LCR, padlock probes, RT-PCR, or multi-probe circularization.Any combination of these methods to target and amplify different locican be used. For example, a combination amplification approach is usedto amplify a genomic DNA locus and an RNA transcript. In one embodiment,a thermostable reverse transcriptase enzyme, such as ThermoScript RT(Lucigen) or GeneAmp Thermostable rTth (Life Technologies), is combinedwith a thermostable DNA polymerase, such as the Stoffel fragment or TaqDNA polymerase. Thermocycling can induce first strand cDNA synthesisfrom the RNA transcript target. Once cDNA from the RNA transcript issynthesized, overlap extension PCR is performed using the cDNA and thegenomic DNA target sequences.

8) Bulk Sequencing Methods

There are a number of new commercial methodologies for polynucleic acidsequencing. These technologies are often referred to as “next generationsequencing,” “massively parallel sequencing,” or “bulk sequencing.”These terms are used interchangeably to describe any sequencing methodthat is capable of acquiring more than one million polynucleic acidsequence tags in a single run. Typically these methods function bymaking highly parallelized measurements, i.e., parallelized screening ofmillions of DNA clones on glass slides. The methods for linking multiplepolynucleic acid targets in single cells could be used in combinationwith any commercialized bulk sequencing method. These methods includereversible terminator chemistry (Illumina), pyrosequencing using polonyemulsion droplets (Roche), single molecule sequencing (PacificBiosciences), and others (IonTorrent, Halcyon, etc.).

After the molecular linkage protocols are performed, and before bulksequencing, it is useful to specifically amplify and purify majoramplicons to reduce the overall sequencing required to obtain usefuldata. Otherwise, many minor amplicons and other kinds of unwantedbackground sequences will be sequenced unnecessarily. This isaccomplished by PCR using only the outer primers and the nucleic acidanalyte obtained from the lysed cells, followed by size selection usinga method such as gel agarose electrophoresis. Other methods, such assize exclusion columns, microfluidic electrophoresis, or microporefilters, might be used to select the proper size molecules.

In one embodiment, the method provides the step of performing a bulksequencing reaction to generate sequence information for at least100,000 fused complexes from at least 10,000 cells within a populationof cells. In another embodiment, the bulk sequencing reaction generatessequence information for at least 75,000, 50,000, or 25,000, or 10,000fused complexes from at least 10,000 cells within a population of cells.

The fused complexes can then be used to quantify the particularbiological or clinical phenomenon of interest. In the case of functionalT or B cell analysis, particular clonotypes that express functionalmolecules can be analyzed by first determining the CDR3 peptide sequenceof the fused complex, and then tabulating the instances of that CDR3peptide linked to a particular effector molecule. In this way the bulksequencing quantifies clonal expansion and biological function of eachsingle clonotype. When primers targeting multiple effector molecules andall possible variable regions are multiplexed into a single assay, andone can separate clonotypes into functional compartments. In the case oflinkage between barcodes and transcript targets, one can stratify thebulk sequencing data by barcode and then tabulate the instances of aparticular barcode linked to a transcript target. When primers targetingmultiple transcripts are multiplexed into a single assay, one can usebarcodes to infer multigenic expression patterns for single cells tracedback to single droplets. In the case of linkage between a mutant orvariable sequence and other mutant or variable sequences, one cananalyze the bulk sequencing data to determine the sequence at each locusin each molecule in the bulk sequencing library, and then tabulate theinstances of each sequence type. If, for example, a mutation in each ofthe two linked targets is required to produce a disease phenotype,quantifying the number of linked targets with two mutations can be usedto detect disease in an individual.

C. Intracellular Linkage in Fixed Cells Followed by Massively ParallelSequencing

The molecular methods described in section B above can be performedintracellularly in thousands to millions of single fixed cells (Embletonet al., 1992 Nucleic Acids Research 20:3831-37; Hviid, 2002 ClinicalChemistry 48:2115-2123; U.S. Pat. No. 5,830,663). The cell membranes ofthe cells serve as reaction compartments, enabling linkage between twoor more genetic loci in thousands to millions of single fixed cellsanalyzed in parallel. Using fixed cells as reaction compartments is morecost-effective than a microfluidic chip to make emulsion microdroplets.Also, heterogeneity in cell size or morphology in a particular cellpopulation is less likely to disrupt the fixed cell method than theemulsion microdroplet method. However, in some cases, leakage of nucleicacids from cells can cause background noise in the molecular geneticanalysis, so care must be taken to wash cells between molecular stepsand perform rigorous quality analysis of analytes. Therefore, in oneembodiment, fixed and permeabilized cells are encapsulated intomicrodroplets, and amplification occurs using fixed, permeabilized cellsin microdroplets instead of lysed cells inside of microdroplets.

1) Molecular Linkage in Fixed Cell Methods

Our work using single cell whole genome amplification (WGA) and PCR fromsingle fixed cells has shown that cell fixation in glutaraldehydeinhibits WGA but not PCR. In any embodiment of intracellular linkageprotocols, care must be taken to ensure that fixation and/orpermeabilization does not inhibit molecular amplification.

For fixation, reagents such as glutaraldehye, paraformaldehyde,IntraStain (Dako), or similar reagents can be used. Forpermeabilization, reagents such as Triton X-100, Tween-20, IntraStain(Dako), or similar reagents can be used (Lippincott-Schwartz 2003 ShortProtocols in Cell Biology; Celis 2005 Cell Biology: A LaboratoryHandbook). After fixation and/or permeabilization, the cells are washedmultiple times in a buffer, such as phosphate-buffered saline (PBS).Once the cells are fixed and/or permeabilized, reaction bufferscontaining primers/probes and enzymes are delivered to the intracellularcompartment without special machinery or methods.

For example, when using RT-PCR to amplify the target loci in singlecells, the fixed and permeabilized cells are soaked in reaction bufferand the first strand cDNA is intracellularly synthesized at 55-70° C.for four hours. Without washing or buffer exchange, one could then usestandard overlap extension PCR thermocycling conditions to amplify andlink the targets. After this amplification procedure, the mixture iswashed several times with PBS, and the supernatant is retained forquality control analysis. The membranes of the resuspended cells arethen disrupted using alkaline lysis buffer or proteinase K solutions(Johnson et al., 2010 Human Reproduction 25:1066-75).

After lysis of the cells and before bulk sequencing, it is useful tospecifically amplify linked complexes to reduce the overall sequencingrequired to obtain useful data. This is accomplished by PCR using onlythe outer primers and the nucleic acid analyte obtained from the lysedcells, followed by size selection using a method such as gel agaroseelectrophoresis.

II. Methods of Pooled Clone Library Deconvolution

Highly multiplexed libraries of nucleic acids are often produced usingparallelized methods that fail to produce individual molecules atoptimized molarity for applications of interest. The method hereinprovides for parallelized synthesis, deconvolution, and re-multiplexingof polynucleic acid libraries. The method retains the advantages of bothparallelized synthesis and individual clone optimization. Thesepolynucleic acid libraries are used for a variety of applications,including but not limited to, multiplexed amplification of targetnucleic acid sequences for sequencing and analysis (FIGS. 15-17).

A. Padlock Probe Synthesis Method

1) Pre-Probe Pool Deconvolution Method

In one embodiment, a pool of thousands of padlock probes that targetsingle nucleotide polymorphisms, or SNPs, are generated. DNAoligonucleotide probe precursors are synthesized in pools (Atactic orNimbleGen). Universal primers are then used to PCR amplifydouble-stranded DNA from the oligonucleotide pool (Porreca et al., 2007Nature Methods 4:931-36). Next, the ends of the double-stranded PCRamplicon library are digested using a restriction enzyme. For example,EcoP15I is used, which cleaves 25 base pairs from the recognition siteand removes the universal PCR binding sites. EcoP15I is one example ofan enzyme that is adequate for subcloning, and uncleaved products do notaffect downstream molecular steps. The digested library is subclonedinto custom-engineered plasmid vectors that confer ampicillinresistance. The plasmids are then transformed into bacterial culturesunder selection with an antibiotic.

FIG. 4 illustrates an example of amplification of a circularizedprobe-target linkage complex (a) in a single cell (b), according to oneembodiment of the invention. Amplification occurs by transformation intobacteria and subsequent selection with antibiotics. The amplicon (a)contains an antibiotic resistant gene and cells (c) that are transformedwith the amplicon are selected in the presence of antibiotics. Cellswithout the circularized probe-target complex (d) are not selected.

2) Single Stranded Probe Synthesis En Masse

In some embodiments, a bacterial stock containing a mixed library ofthousands of clones, each targeting a particular SNP, is used for singlestranded probe synthesis en masse. For example, the bacterial culturesare spread on LB agar plates under ampicillin selection, and thenindividual colonies are picked. Next, PCR with barcoded primers is usedto amplify the probe sequence and flanking universal priming regions.The result is an amplicon that contains both the probe sequence and abarcode that can be traced back to a single well. In one embodiment, aunique molecular barcode will indicate a particular well position in aparticular 384-well plate. For example, the system could have 3,840unique barcodes that indicate the well positions and plate number for3,840 PCRs in one of ten 384-well plates. To deconvolute a 10,000-plexlibrary of clones, four rounds of deconvolution are performed using theset of 3,840 barcoded PCRs, and oversampling and screening a total of15,360 clones. For each round of deconvolution, the PCR products canthen be pooled and sequenced using any bulk sequencing method.

With the probe sequences matched to a barcode, a deconvolution algorithmcan then be used to deconvolute the library. Because the barcode ismatched to the insert sequence, a table is created that matches thebarcode sequence to the original well and plate, and accordingly, thismatches the insert sequence to a well. The bacterial clones can then bestored as glycerol stocks, and sequences of these stocks can then becatalogued in a database and stored at −80° C.

To synthesize single stranded padlock probes from the template glycerolstocks, a derivation of the SMART technique is used (Krishnakumar etal., 2008 PNAS 105:9296-9301). At a high level, this method involves (i)digestion of a double stranded DNA with a restriction endonuclease; (ii)dephosphorylation of the “sticky end”; (iii) digestion of the second endof the double stranded DNA with a second restriction endonuclease; and(iv) digestion of the desphosphorylated strand of DNA using a λexonuclease. First, the desired clones are picked, and then cultured in384-well plates. After incubation overnight, the optical density of eachculture is assessed, and then the stocks are equalized. 5 μL from thenormalized bacterial cultures is pooled, and the plasmid pool ispurified using standard methods (Qiagen). Next, a set of universal PCRprimers is used to generate a pool of double-stranded PCR amplicons. Theresulting PCR mixture is then subjected to digestion with a restrictionenzyme, such as HaeIII (NEB), followed by dephosphorylation with shrimpalkaline phosphatase (SAP). After desphosphorylation, the analyte isdigested with a restriction enzyme, such as BstUI (NEB). This productcan then be digested with X exonuclease (NEB), producing single strandedDNA molecules. Finally, single stranded DNA (ssDNA) is purified from anyundigested double stranded DNA using a commercial kit (Zymo Research).In this way, hundreds of thousands of probes are synthesized inparallel.

B. Cell Clone Deconvolution

The methods in Section II. A. can also be used to deconvolute mixedlibraries of cells or organisms with different underlying geneticcharacteristics. The goal is to separate the mixed library of clonesinto reaction compartments, perform barcoded PCR followed by bulksequencing on the clones, and then map sequence data back to the clonesin reaction compartments. In one example, a population of mammaliancells is mutagenized and then clonal populations of mutagenized cellsare isolated from the mixed population. In this embodiment, singlemutagenized cells are sorted into reaction compartments, and thentargeted barcoded PCR or padlock probes are performed at genetic loci ofinterest. Bulk sequencing data is used to trace back to the originalclones, and then the physical clone stocks is used for furtherinvestigation or use.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3^(rd) Ed.(Plenum Press) Vols A and B (1992).

Example 1 Methods of T-Cell Analysis

The immune system responds to disease by inducing cellular responses.Nearly all immunology is involved with detection of clonotype expansionor contraction in response to an antigen and/or functional analysis ofthe expanded or contracted clonotypes. Described in this example aremethods that leverage the information contained in immune response todiagnose and treat disease. Active and/or memory cells are particularlyinformative because these cells indicate a functional immune response toa disease, and therefore have high information content. Variable DNAregions and RNA transcripts were analyzed in single cells frompopulations of activated and/or memory immune cells, and then correlatedwith disease. These profiles were used to develop noninvasivediagnostics, high-value diagnostics that inform treatment regimens, andnovel therapeutic agents.

T cells include T cell receptors (TCR) that recognize antigens andcontrol immune responses. The T cell receptor is composed of twosubunits: α and β or γ and δ. Current methods to examine T cells bytheir T cell receptors overwhelmingly sequence T cell receptor subunitsfrom bulk populations that range from a few to millions of cells. Thisresults in a catalogue of subunit sequences (α or δ) that are unlinkedto the other corresponding subunit sequence found in individual cells (βor γ). This gives population level information about T cell receptordiversity but does not give a description of individual T cell receptorsin individual cells by both subunits (α and β or γ and δ). By linkingsequences in a single cell using the methods in Sections I. A-C, theTCRs of individual cells in mixed populations are analyzed with finerresolution, and this allows an unprecedented mapping of human T-celldiversity.

The sequences of TCR subunits and immune functionality molecules werelinked using the methods described in Sections I. A-C. This approach,called “functional T cell sequencing,” focused specifically on T cellslikely to have a clinically or biologically relevant function. Forexample, the immune function of a T cell is indicated by expression ofboth clonal TCR and signaling molecules such as interleukin-4 (IL-4).Naïve T cells express clonal TCR but do not express signaling moleculessuch as IL-4, and have different immune functions. The TCR was linked tothe signaling molecule, which in turn linked the TCR to clinicalfunction. Primers amplifying the full TCRβ repertoire were linked to asingle immune effector molecule, such as IL-4. Primers amplifying thefull TCRβ repertoire were linked to dozens of immune effector molecules,resulting in a full T cell phenotype for each T cell clonotype in theassay.

Examples of molecules that are associated with immune function and thatare linked to a TCR sequence include, but are not limited to:interleukin-2 (IL-2), interleukin-4 (IL-4), interferon gamma (IFNγ),interleukin-10 (IL-10), interleukin-1 (IL-1), interleukin-13 (IL-13),interleukin-17 (IL-17), interleukin-18 (IL-18), tumor necrosis factoralpha (TNFα), tumor necrosis factor beta (TNFβ), T-box transcriptionfactor 21 (TBX21), forkhead box P3 (FOXP3), cluster of differentiation 4(CD4), cluster of differentiation 8 (CD8), cluster of differentiation 1d(CD1d), cluster of differentiation 161 (CD161), cluster ofdifferentiation 3 (CD3), and T-box transcription factor TBX21 (T-BET).

The TCR β chain was linked to a molecule associated with immunefunction. In another exemplary method, the TCR α and β, or TCR γ and δ,or any of the individual subunits, were linked to immune functionalitymolecules. Published primers optimized for amplification of recombinedgenomic TCR were used (Robins et al., 2009 Blood 114:4099-107). Much ofthe peptide variability of the TCR was encoded in CDR313, which wasformed by recombination between noncontiguous variable (V), diversity(D), and joining (J) segments in the b chain loci (Wang et al., 2010PNAS 107:1518-23). Previously published PCR primers targeting the CDR3βlocus can also be used (Robins et al., 2009 Blood 114:4099-107; Robinset al., 2010 Science Translational Med 2:47ra64). This set of forty-fiveforward primers and thirteen reverse primers amplify the ˜200 base pairrecombined genomic CDR3β region for multiplex amplification of the fullCDR3β complement of a sample of human peripheral blood mononuclearcells. The CDR3β region begins with the second conserved cysteine in the3′ region of the Vβ segment and ends with the conserved phenylalanineencoded by the 5′ region of the Jβ segment (Monod et al., 2004Bioinformatics 20:i379-i385). Thus, amplified sequences wereinformatically translated to locate the conserved cysteine, obtain theintervening peptide sequence, and tabulate counts of each unique clonein the sample.

Examples of primers that can be used for multiplex amplification of TCRsequences and linkage to various immune effector molecules are shown inTable 2. These primers have been used, for example with the methods ofSection I. A-C, to amplify and link TCR sequences to various immuneeffector molecules.

Example 2 High-throughput Protocol for TCRβ Repertoire LibraryConstruction

In one embodiment, a high-throughput protocol was implemented for humanor mouse TCRβ repertoire library construction. The libraries weresequenced directly on the GAIIx next-gen sequencing platform (Illumina)For human samples, multiplex PCR was performed using a set of 20 primersto amplify across all 50 V segments and 10 primers to amplify across all13 J segments. The primers libraries generated libraries that were thereverse complement of the native TCRβ sequence. This enabled sequencingfrom the J side of the constructs without further manipulation. Theprimers also had tails with the same sequence as a portion of theIllumina TruSeq library adapter. The 30 primers were pooled in a single400 μl PCR, which contained genomic DNA from at least 5×10⁵ cells. Thereactions were then thermocycled for no more than 25 cycles, dependingon the number of input cells. After thermocycling, a PCR column (Qiagen)was used to remove the primers. Next, a second round of PCR wasperformed, using an aliquot of the purified first round analyte and aset of universal primers. The universal primers for the second round ofPCR annealed to the tails of the first primers, producing final PCRproducts that had the full Illumina sequencing adapter sequence fused toa library of TCRβ sequences. The universal primers also had barcodetags, which enabled multiplexing of dozens of samples in a singlenext-generation sequencing lane. Finally, the libraries were purifiedwith gel size selection, and quantified with a quantitative PCR kit(Kapa Biosystems) prior to sequencing. Over 300 TCRβ libraries werebuilt and sequenced using this protocol.

FIG. 31 shows a simplified workflow for high-throughput generation ofTCRβ repertoire libraries. The first round used a set of 30 primers toamplify the full TCRβ repertoire and attaches universal priming regions.The second round amplified the repertoire with universal primers andadded sequences for next-generation sequencing.

Example 3 Protocol Optimization Using 48-plex Pool of TCRβ PlasmidClones

The true content of any particular TCRβ repertoire is not known, so anendogenous TCRβ repertoire cannot serve as a gold standard for protocoloptimization. A 48-plex pool of mouse TCRβ plasmid clones was designedto act as template for protocol optimization. First, multiplexedamplification was performed of the mouse TCRβ repertoire as described inExample 2. The PCR products were subcloned using the TOPO-TA vector(Life Technologies), transformed post ligation into TOP10 competentcells (Life Technologies), and 48 transformed colonies were picked.Next, the clones were sequenced by Sanger sequencing to identify theTCRβ clonotype sequences. All of the clones were unique, and representeda broad range of possible V-Jβ combinations. The plasmids were thenmixed in a single tube, across three orders of magnitude and with sixreplicates at each concentration.

The 48-plex mixture was used to optimize the TCRβ amplificationprotocol. The purification methodology after the first and second PCRsteps, the number of cycles in the first PCR, and the annealingtemperature in the first PCR were optimized. WA PCR column or gelexcision for the purification technology were used. Due to spuriousmispriming, the first round of PCR produced multiple bands in additionto a major band in the target size range of 150-200 bp. Gel excisionremoved the undesired material, but the process was tedious and resultsin loss of up to 75% of the desired material. Protocols with fewer firstPCR amplification cycles typically produce less severe amplificationbias, whereas amplification bias is typically skewed in protocolswith >30 cycles. Annealing temperature controls the stringency ofpriming events, with lower temperatures producing higher yields but lessspecificity.

68 Illumina libraries were constructed using the mixture of 48 plasmidsand varying protocol parameters as described above. The libraries weresequenced on a next-generation sequencing machine (Illumina) toobtain >500 k paired-end 80 bp sequence tags for each library. Toanalyze the sequencing data, each 2+80 by sequence tag was aligned tothe sequences of the 48 known clonotypes to obtain the best match. Thenumber of tags aligned to each plasmid for each library was counted, andthen these results were correlated with the expected ratios of the inputplasmid clones. A linear regression analysis to fit each data set wasperformed (see Table 1: yielding correlation, R² of 1, and a slope of 1.The protocol used 15 cycles of amplification for the first PCR, anannealing temperature of 61° C., PCR column purification after the firstPCR, and gel purification following the second PCR.

TABLE 1 Analysis of selected pilot protocol optimization experiments. R²and slope were computed from a regression analysis between the observedcount of sequences in each library versus the known input count.Conditions in row 3 (bold) are an example of an optimized protocol. 1stPCR 1st PCR 2nd PCR Cycles 1st PCR Ta Cleanup Cleanup R2 Slope 15 57column gel 0.56 0.54 15 59 column gel 0.7 0.68 15 61 column gel 0.720.71 15 63 column gel 0.69 0.7 25 57 column gel 0.47 0.43 25 59 columngel 0.44 0.4 25 61 column gel 0.45 0.45 25 63 column gel 0.41 0.39 35 57column gel 0.47 0.41 35 59 column gel 0.43 0.37 35 61 column gel 0.420.4 35 63 column gel 0.41 0.4

Example 4 TCRβ Repertoire Data Analysis

Because the TCRβ repertoire contains as many as 5×10⁶ clonotypes, andCDR3 regions often differ by only a few nucleotides, a sophisticatedcustom analysis platform was necessary just to identify the clones inthe library. The turnkey fast-alignment methods, such as BLAST (Altschulet al., 1990), BLAT (Kent 2002), and SOAP (Li et al., 2008), wereinadequate for the task at hand, because they resulted in many spuriousmatches. Moreover, highly accurate turnkey methods such asSmith-Waterman (Smith and Waterman, 1981) were cumbersomely slow forthis kind of analysis. Finally, all of these methods would require ahuge reference library (10¹⁵ diversity) of all possible CDR3 nucleotidesequences, which is a computational burden.

To address these problems, an algorithm was built that is faster thanany current method by almost an order of magnitude, and which has thesame accuracy as standard alignment methods. A table of 4-8 nucleotide“words” that uniquely identify the V and J segments of mouse or humanwithin the amplified region is generated. The validity of each match istested by identifying the distance to and the sequence of the secondconserved cysteine. The match was accepted as correct only if bothdistance and sequence confirm the match. Using data from our TCRβrepertoire sequencing experiments, we typically identified ˜99.98% ofV-Jβ combinations unambiguously. The remaining reads were discarded.

We also employed two further quality control steps: (i) the CDR3 regionmust not contain any sequencing errors in the form of uncalled bases;and (ii) the CDR3 region is in frame as defined by the second conservedcysteine. If all quality tests are passed, the method identified theprotein coding sequence of the CDR3 region within the known readingframe for that particular gene. This algorithm ensured speed, accuracyand lowest error rates. It can easily be adapted for use with othervariable gene families, such as TCRα, or IgH.

A number of experiments were performed to demonstrate the utility of ourprotocols for deep TCRβ sequencing. Mouse bone marrow transplantationswere performed in matched and mismatched genetic backgrounds. Todetermine the systemic impact of these transplantation events on themice, the T cell repertoires of the colon were examined. The most commonTCRβ clonotypes in colons from replicate mismatched bone marrowtransplantations were more closely related than the most common TCRclonotypes in a colon from syngenic transplantation, especially in thetop 1% of clones. Profiles of control colons were nearly identical inthe top 1% of clonotypes. These data indicate that the protocolsdescribed herein produce quality, quantitative data of utility toresearch customers.

Example 5 Constructing a Control Library of TCRI Clones and OptimizingPCR Conditions Using the Control Library

Additional experiments are performed to build a library of 960 TCRβclones that contains at least one representative from each of the 650possible human V-Jβ combinations. This set of clones is used formolecular and statistical optimizations. A plasmid library of human TCRβis generated as described above in Example 4. About 3,000 transformantcolonies are picked and the clones are sequenced using standardcapillary sequencing (e.g., Sequetech). The V-Jβ pairing correspondingto each sequenced clone is identified as described above in Example 4.The goal is to obtain at least one representative clone for each V-Jβpair. If sequencing finds that some V-Jβ pairs are missing, those pairsare rescued by making libraries of TCRβ using only primers for thosemissing V-Jβ pairs, subcloning, and sequencing. After several rounds,clones are identified for every possible V-Jβ pair. These plasmids aremixed into a single template mixture, with 96 clones at eachconcentration and 10 different concentrations across three orders ofmagnitude.

Example 6 Optimizing PCR Conditions Using the Control Library

Previous experiments have shown that the first PCR amplification causesmost of the amplification bias. Additional experiments are performedusing the 960-clone pool and next-generation sequencing to furtheroptimize first PCR cycle number. About 60 TCRβ libraries are generatedfrom the plasmid mixture, with four replicates for each of the 15 cyclenumbers between 10 and 25. The library mixtures are quantified and ˜4million sequences are obtained from each library a GAIIx next-gensequencer (Illumina). The V-Jβ pairing corresponding to each sequencedclone as described above in Example 4, and the counts of sequence tagsare tallied for each clone in each data set.

Prior work has shown that GC content can affect amplification efficiency(Markoulatos et al., 2002). The immense variety of V(D)Jβ combinationsresult in an assortment GC contents and lengths. The amplification biasis tested after addition of various reagents, such as betaine ormagnesium chloride. Approximately 60 TCRβ libraries are generated fromthe plasmid mixture, with four replicates for each of 15 differentbuffers. The library mixtures are quantified and ˜4 million sequencesare obtained from each library using a GAIIx next-gen sequencer(Illumina) The V-Jβ pairing is identified corresponding to eachsequenced clone as described above in Example 4, and the counts ofsequence tags are tabulated for each clone in each data set.

Example 7 T Cell Analysis and Transplant Monitoring

Methods of the invention are applied to post-transplant immunemonitoring. After an allogeneic transplant (i.e., kidney or liver), ahost's T cells response to transplants are assessed to monitor thehealth of the host and the graft. Molecular monitoring of blood or urineis helpful to detect acute or chronic rejection before a biopsy wouldtypically be indicated. For example, detection of alloantibodies tohuman leukocyte antigen (HLA) has been associated with chronic allograftrejection (Terasaki and Ozawa, 2004 American Journal of Transplantation4:438-43). Other molecular markers include b₂-microglobulin, neopterin,and proinflammatory cytokines in urine and blood (Sabek et al., 2002Transplantation 74:701-7; Tatapudi et al., 2004 Kidney International65:2390; Matz et al., 2006 Kidney International 69:1683; Bestard et al.,2010 Current Opinion in Organ Transplantation 15:467-473). However, noneof these methods has become widely adopted in clinical practice, perhapsdue to low specificity and sensitivity. Prior work has shown thatregulatory T cells (Treg) induce graft tolerance by down-regulatinghelper T cells (Th) (Graca et al., 2002 Journal of Experimental Medicine195: 1641). Additionally, transplanting hematopoietic stem cells fromHLA-mismatched donors into the recipient has resulted in long-termnonimmunosuppressive renal transplant tolerance up to 5 years aftertransplant (Kawai et al., 2008 NEJM 358:353-61).

Primers are designed that target transcripts from several immunefunctionality genes (described above), which produce overlap extensionfusion constructs with CDR3β amplicons. In one embodiment, these primersare designed to specifically amplify cDNA by spanning RNA splicejunctions and hybridize to cDNA from processed messenger RNA. Examplesof molecules that are associated with immune function include, but arenot limited to, T-BET and IFN-g, which indicate T helper 1 cells (Th1);GATA3 and IL-4, which indicate T helper 2 cells (Th2); IL-17, whichindicates T helper 17 cells (Th17); and FoxP3 and IL-10, which indicateT regulatory cells (Treg). Such signaling molecules are members of largeprotein families with strong homology between paralogues, which mayresult in background amplification during PCR. Accordingly, nucleotidealignments of all of the paralogues in each family (i.e., all of theinterleukin genes) are generated and PCR primers are designed that spanexons and have the lowest possible sequence homology to other genes inthe family

Functional T cell monitoring involves the following steps: (i) isolationof single peripheral blood mononuclear cells in emulsion microdropletreactors; (ii) overlap extension amplification of complexes between TCRβand immune functionality molecules in microdroplet reactors; and (iii)emulsion reversal followed by bulk sequencing. The TCRβ and immunefunctionality primer sets will be combined to produce major ampliconfusion constructs from the minor amplicons. The overlap extensionprimers are a combination of the reverse TCRβ primers with approximatelyhalf of each immune functionality molecule forward primer, which resultsin a total of 91 fusion reverse TCRβ primers. The fusion primers betweenthe forward primer for each immune functionality minor amplicon containapproximately half of each of the 13 TCRβ reverse primers, for a totalof 91 fusion reverse immune functionality primers. The final result isthat the overlap between any pair of TCRβ and immune functionality minoramplicons has a melting temperature of approximately 55-65° C., suchthat each minor amplicon acts as a primer for the paired amplicon. Inthe final reaction mixtures, the outer primers are diluted to a finalconcentration of 0.1 μM, and the inner primers are diluted to 0.01 μM,such that the inner primers are limiting reagents.

Example 8 T Cell Analysis and Latent Tuberculosis Diagnosis

Latent tuberculosis (TB) is a major global epidemic, affecting as manyas 2 billion people worldwide. There is currently no reliable test forclinical diagnosis of latent TB. This technology gap has severe clinicalconsequences, since reactivated TB is the only reliable hallmark oflatent TB. Furthermore, clinical trials for vaccines and therapies lackbiomarkers for latent TB, and therefore must follow cohorts over manyyears to prove efficacy.

The major current vaccine for tuberculosis, bacillus Calmette-Guérin(BCG), is an unreliable prophylactic. In a meta-analysis of dozens ofepidemiological studies, the overall effect of BCG was 50% against TBinfections, 78% against pulmonary TB, 64% against TB meningitis, and 71%against death due to TB infection (Colditz et al., 1994 JAMA271:698-702). Additionally, the rapid rise in multidrug resistant TB hasincreased the need for new vaccine and immunotherapy approaches. Up to90% of infected, immunocompetent individuals never progress to disease,resulting in the huge global latent TB reservoir (Kaufmann, 2005 Trendsin Immunology 26:660-67).

Since tuberculosis is a facultative intracellular pathogen, immunity isalmost entirely mediated through T cells. Interferon-g expressing Thelper 1 (Th1) cells elicit primary TB response, with some involvementby T helper 2 cells (Th2). After primary response, the bacteria becomelatent, controlled by regulatory T cell (Treg) and memory T cells(Tmem). Recently, eleven new vaccine candidates have entered clinicaltrials (Kaufmann, 2005 Trends in Immunology 26:660-67). These vaccinesare all “post-exposure” vaccines, i.e., they target T cell responses tolatent TB and are intended to prevent disease reactivation. Because ofthe partial failure of BCG to induce full immunity, rational design andvalidation of future TB vaccines should include systematic analysis ofthe specific immune response to both TB and the new vaccines.

For decades, the standard of care for diagnosis of latent tuberculosishas been the tuberculin skin test (TST) (Pai et al., 2004 LancetInfectious Disease 4:761-76). More recently, two commercial in vitrointerferon-g assays have been developed: the QuantiFERON-TB assay andthe T SPOT-TB assay. These assays measure cell-mediated immunity byquantifying interferon-g released from T cells when challenged with acocktail of tuberculosis antigens. Unfortunately, neither the TST northe newer interferon-g tests is effective at distinguishing latent fromcleared TB (Diel et al., 2007 American Journal of Respir Crit Care Med177:1164-70). This is a significant problem because patients withoutclinical evidence of latent TB (i.e., visualization of granulomas) butwith positive TST or interferon-g test typically receive 6-9 months ofisoniazide therapy, even though this empiric intervention is unnecessaryin patients who have cleared primary infection and can cause seriouscomplications such as liver failure.

Prior work has demonstrated that T cell responses are used todistinguish latent from active TB (Schuck et al., 2009 PLoS One4:e5590). The premise of this prior work is that immune cells directedagainst TB antigens will be expanded in the memory T cell population ifthe TB is latent, but expanded in a helper T cell fraction if the TB isactive. Functional T cell sequencing is used to distinguish latent TBfrom cleared TB. The protocol involves: (i) capture of single T cells inemulsion microdroplets; (ii) microdroplet reverse transcription andamplification at target loci; (iii) microdroplet synthesis of fusioncomplexes between two or more target loci; and (iv) reversing emulsionsand sequencing major amplicons with bulk sequencing. Sequence specificPCR is used after overlap extension RT-PCR to detect the presence of aparticular biomarker for latent TB.

Example 9 T Cell Analysis and Diagnosing or Monitoring Disease

Similarly, functional T cell monitoring is used for diagnosis andmonitoring of nearly any human disease. These diseases, include but arenot limited, to systemic lupus erythmatosis (SLE), allergy, autoimmunedisease, heart transplants, liver transplants, bone marrow transplants,lung transplants, solid tumors, liquid tumors, myelodysplastic syndrome(MDS), chronic infection, acute infection, hepatitis, human papillomavirus (HPV), herpes simplex virus, cytomegalovirus (CMV), and humanimmunodeficiency virus (HIV). Such monitoring includes individualdiagnosis and monitoring or population monitoring for epidemiologicalstudies.

T cell monitoring is used for research purposes using any non-humanmodel system, such as zebrafish, mouse, rat, or rabbit. T cellmonitoring also is used for research purposes using any human modelsystem, such as primary T cell lines or immortal T cell lines.

Example 10 B Cell Analysis

Antibodies are produced by recombined genomic immunoglobulin (Ig)sequences in B lineage cells. Immunoglobulin light chains are derivedfrom either κ or λ genes. The λ genes are comprised of four constant (C)region genes and approximately thirty variable (V) region genes. Incontrast, the icgenes are comprised of one C region gene and 250 Vregion genes. The heavy chain gene family is comprised of severalhundred V gene segments, fifteen D gene segments, and four joining (J)gene segments. Somatic recombination during B cell differentiationrandomly chooses one V-D-J combination in the heavy chain and one V-Jcombination in either κ or λ light chain. Because there are so many genesegments, millions of unique combinations are possible. The V regionsalso undergo somatic hypermutation after recombination, generatingfurther diversity. Despite this underlying complexity, it is possible touse dozens of primers targeting conserved sequences to sequence the fullheavy and light chain complement in several multiplexed reactions (vanDongen et al., 2003 Leukemia 17: 2257-2317).

Any of the individual immunoglobulin subunits are linked to immunefunctionality molecules that indicate B cell activity or subpopulations.A first target nucleic sequence, a second target nucleic acid sequenceor both target nucleic acid sequences can comprise an immunoglobulinsequence. Alternatively the first target nucleic acid sequence cancomprise an immunoglobulin sequence, and the second sequence cancomprise a second molecule associated with immune cell function.Examples of functional B cell marker molecules include, but are notlimited to, major histocompatibility complex (MHC), cluster ofdifferentiation 19 (CD19), interleukin 7 receptor (IL-17 receptor),cluster of differentiation 10 (CD10), cluster of differentiation 20(CD20), cluster of differentiation 22 (CD22), cluster of differentiation34 (CD34), cluster of differentiation 27 (CD27), cluster ofdifferentiation 5 (CD5), and cluster of differentiation 45 (CD45),cluster of differentiation 38 (CD38), cluster of differentiation 78(CD78), interleukin-6 receptor, Interferon regulatory factor 4 (IRF4),and cluster of differentiation 138 (CD138). A primer pool that amplifiesthe full IgH complement of B cells is combined with a single B cellmarker primer pair. This assays all of the B cell clonotypes in aparticular functional group, such as Bmem. Alternatively, a primer poolthat amplifies the full IgH complement of B cells is combined withdozens of B cell marker primer pairs. This assay provides the fullphenotype for each clonotype in the cell mixture.

A method is provided for linking IgH and Igκ: IgH and Igλ are linked insingle cells to immune functionality molecules that indicate B cellactivity or subpopulations. The vast majority of diversity in the B cellrepertoire is comprised of the V-D-J regions of IgH and V-J regions ofIgκ (Sandberg et al., 2005 Journal of Molecular Diagnostics 7:495-503;Boyd et al., 2009 Science Translational Med 1:12ra23).Previously-reported primer pools (van Dongen et al., 2003 Leukemia 17:2257-2317) are used to amplify these regions of IgH and Igκ Five primerpools in separate reactions are used to amplify the IgH and Igκcomplement of a healthy human. The amplified material sequenced withbulk sequencing. To analyze the bulk sequencing results, the IgBLASTalgorithm and database is used to determine the V-D and D-J junctions ofIgH and align the IgH and Igκ sequences to germ line gene segments.Overall, this method is more highly parallelized thanpreviously-reported methods for single cell Ig analysis (U.S. Pat. No.7,749,697).

Example 11 B Cell Analysis and Drug Discovery

Antibody therapeutics are increasingly used by pharmaceutical companiesto treat intractable diseases such as cancer (Carter 2006 Nature ReviewsImmunology 6:343-357). However, the process of antibody drug discoveryis expensive and tedious, requiring the identification of an antigen,and then the isolation and production of monoclonal antibodies withactivity against the antigen. Individuals that have been exposed todisease produce antibodies against antigens associated with thatdisease, so it is possible mine patient immune repertoires forantibodies that could be used for pharmaceutical development. However, afunctional monoclonal antibody requires both heavy and light chainimmunoglobulins. Overlap extension PCR and/or overlap extension RT-PCRin single cell emulsion microdroplets is used to capture functionalantibody sequences from patient B cell repertoires. Briefly, the methodinvolves the following steps: (i) isolation of single B cells inaqueous-in-oil microreactors using a microfluidic device; (ii) molecularlinkage between heavy and light chain immunoglobulin (IgH and Igκ)amplicons inside the single cell microreactors; and (iii) reversal ofthe emulsions followed by bulk sequencing of the linked polynucleic acidsequences. This produces heavy and light chain pairings from millions ofsingle B cells analyzed in parallel, which are mined as potentialtherapeutic agents.

The fusion primer sequences for overlap extension PCR and overlapextension RT-PCR are identical to the independent IgH and Igκ primers,except certain primers contain additional polynucleotide sequences foroverlap extension: (i) the forward primer of the IgH locus has a random10-20nt sequence with no complementarity to either target; (ii) thereverse primer of the IgH loci has a 10-20nt sequence withcomplementarity to the forward primer of Igκ, and (iii) the forwardprimer of Igκ has complementarity to the reverse primers for the IgHlocus. In the final reaction mixtures, the outer primers are diluted toa final concentration of 0.1 μM, and the inner primers are diluted to0.01 μM, such that the inner primers will be a limiting reagent. Thisdrives formation of the major amplicon.

Example 12 B Cell Analysis and Monitoring Immunity

Humoral memory B cells (Bmem) help mammalian immune systems retaincertain kinds of immunity. After exposure to an antigen and expansion ofantibody-producing cells, Bmem cells survive for many years andcontribute to the secondary immune response upon re-introduction of anantigen. Such immunity is typically measured in a cellular orantibody-based in vitro assay. In some cases, it is beneficial to detectimmunity by amplifying, linking, and detecting IgH and light chainimmunoglobulin variable regions in single B cells. Such a method is morespecific and sensitive than current methods. Massively parallel B cellrepertoire sequencing is used as described in Example 13 to screen forBmem cells that contain a certain heavy and light chain pairing which isindicative of immunity. In another exemplary method, single cell heavyand light chain pairing are combined with functional B cell sequencing,i.e., developing overlap extension RT-PCR primers that target RNAtranscripts that are overrepresented in Bmem cells (i.e., CD27). Bycombining light and heavy immunoglobulin amplification with geneexpression of Bmem or plasma cell immune function transcripts, sortingcells by FACS or other tedious methods are avoided.

Example 13 B Cell Analysis and Diagnosing and Monitoring Disease

B cell monitoring is used for diagnosis and monitoring of nearly anyhuman disease. These diseases include, but are not limited to, systemiclupus erythmatosis (SLE), allergy, autoimmune disease, hearttransplants, liver transplants, bone marrow transplants, lungtransplants, solid tumors, liquid tumors, myelodysplastic syndrome(MDS), chronic infection, acute infection, hepatitis, human papillomavirus (HPV), herpes simplex virus (HSV), cytomegalovirus (CMV), andhuman immunodeficiency virus (HIV). Such monitoring could includeindividual diagnosis and monitoring or population monitoring forepidemiological studies.

B cell monitoring is also used for research purposes using any non-humanmodel system, such as zebrafish, mouse, rat, or rabbit. B cellmonitoring is used for research purposes using any human model system,such as primary B cell lines or immortal B cell lines.

Example 14 Methods for Noninvasive Prenatal Diagnosis

In the absence of prenatal diagnosis, approximately 2% of babies haveserious physical or mental handicaps, approximately 3.3% of babies havesome form of congenital malformation, and approximately 0.5% have aphenotypically-significant chromosome abnormality. Current clinicalmethods for prenatal diagnosis are invasive and carry significant risksto the fetus, restricting their use to patients of advanced maternalage. Noninvasive, accurate technologies are needed for first trimesterprenatal genetic diagnosis. Most current preclinical methods fornoninvasive prenatal diagnosis capture and diagnose circulating fetalcells. These methods rely on cell surface proteins and/or cellmorphology to enrich for particular populations of fetal cells. Suchflawed approaches have failed to reach the clinic despite decades ofintense research and development.

Isolation of circulating fetal nucleated red blood cells (FNRBCs) frommaternal blood is one approach to noninvasive prenatal diagnosis.Nucleated red blood cells are among the first hematopoietic cell typesproduced during fetal development. These cells cross the placenta andare detectable at low concentrations in maternal blood during the firsttrimester (Ganshirt et al., 1994 Lancet 343:1038-9). Another attractivefeature of FNRBCs is their short lifespan compared to other circulatingfetal cell types (Pearson, 1967 Journal of Pediatrics 70:166-71), makingthem unlikely to persist in maternal blood from previous pregnancies.

The scarcity of circulating fetal cells, estimated at one fetal cell per10⁵-10⁹ maternal cells (Price et al., 1991 Am J Obstet Gynecol165:1731-7; Ganshirt-Ahlert et al., 1994 Clin Genet 38:38-43),necessitates the use of sensitive and specific fetal cell enrichmentmethods prior to diagnosis. Widely-adopted enrichment methods includecombinations of density gradient centrifugation (Samura et al., 2000Prenat Diagn 20:281-6), fluorescence activating cell sorting (FACS), andmagnetic cell sorting (MACS) (Busch et al., 1994 Ann NY Acad Sci 731:144-6). Despite the development of these methods, none have beencommercialized.

i. Methods for Noninvasive Prenatal Diagnosis of Single Gene Disorders

LCR or padlock probes are used to capture and amplify paternal-specificalleles in an allele-specific manner and to perform overlap extensionPCR to detect disease alleles (FIGS. 26-30). The method involves thefollowing steps: (i) parental genotyping to find paternal-specificpolymorphisms; (ii) isolation of single mononuclear cells from maternalblood into emulsion microdroplets; (iii) amplification of disease andpaternal-specific “linker” loci by a modified LCR/PCR protocol inemulsion microdroplet reactors; (iv) overlap extension amplification ofcomplexes between disease and linker loci in microdroplet reactors; (v)recovery of linked complexes by emulsion reversal; and (vi) massivelyparallel sequencing. The massively parallel sequencing data are analyzedto quantify instances of linked genotypes. Only microdroplet reactorsthat contain single fetal cells yield linked complexes between thedisease locus and the paternal-specific allele. Both alleles amplifyfrom the fetal cell, providing the physician with status as a carrier,homozygous normal, or homozygous affected.

LCR probes are designed to target a locus associated with a disease anda linker SNP locus. The LCR probes are 20-30 nucleotides long and havemelting temperatures (Tm) of approximately 55-65° C. The 5′ nucleotidesare phosphorylated, and probes are designed to minimize probeself-complementarity, as well as complementarity between probes. Inaddition to regions of complementarity to target loci, three of theprobes include polynucleotide sequences that enable amplification afterligation: (i) the 5′ probe for the disease locus have a random 10-20ntsequence with no complementarity to either target locus; (ii) the 3′probe for the disease locus has a 10-20 nucleotide sequence withcomplementarity to the 5′ end of the linker SNP locus; and (iii) the 5′probe for the linker SNP locus have complementarity to the 3′ end of thedisease locus (FIGS. 26-30).

For each disease and linker locus pair, a reaction mixture is formulatedusing cell line genomic DNA, the LCR probes, the PCR primers, Ampligase(Epicentre), Stoffel fragment DNA polymerase (Life Technologies), andreaction buffer (after Hardenbol et al., 2005; 20 mM Tris-HCl, 25 mMKCl, 10 mM MgCl₂, 0.5 mM NAD, 0.01% Triton X-100). The “inner” probesare added at 1/10 of the concentration of the other oligonucleotides inthe reaction. For the initial annealing and ligation, the mixtures areincubated for 4 minutes at 20° C., 5 minutes at 95° C., and 15 minutesat 60° C. Then, standard PCR thermocycling conditions are used toamplify the minor and major amplicons (e.g., 95° C., 5 minutes; [95° C.,30 seconds; 60° C., 30 seconds; 72° C., 30 seconds]×30 cycles).

After bulk sequencing of the major amplicons, disease and unaffectedalleles are analyzed to diagnose the fetus as homozygous normal,heterozygous carriers, or homozygous affected. In heterozygous carriers,major amplicons linked to the paternal-specific allele compriseapproximately 50% disease alleles and 50% normal alleles. Similarly, inhomozygous carriers, major amplicons linked to the paternal-specificallele comprise of nearly 100% disease alleles. This method can beextended beyond single nucleotide mutations to find paternal-allelespecific gene expression patterns and/or multiplexed analysis of manygermline mutations in circulating fetal cells.

Examples of genes that are often mutated and are of interest in prenataldiagnostics include, but are not limited to, cystic fibrosistransmembrane receptor (CFTR), aspartoacylase (ASPA), Fanconi anemia,complementation group C (FANCC), Glucose-6-phosphatase (G6CP),Glucocerebrosidase (GBA), Hexosaminidase A (HEXA), hemoglobin beta(HBB), Frataxin (FXN), low density lipoprotein receptor (LDLR), andmethyl CpG binding protein 2 (MECP2).

For example, in FIG. 26, single cell sequence linkage by ligase chainreaction combined with overlap extension polymerase chain reaction isillustrated, as applied to a method for noninvasive prenatal diagnosis.The target nucleic acid (g) is a paternal-specific allele, the targetnucleic acid (h) is a first disease allele, and the target nucleic acid(i) is a second disease allele. Notably, both alleles (h) and (i) areamplified in any cell (j) that contains the paternal-specific variant,and no major amplicons are produced in cells that lack thepaternal-specific nucleotide variant. Primer (a) is a forward LCR probeand primer (b) is a reverse LCR probe for amplifying target nucleic acid(g). Primer (e) is a forward PCR primer and primer (f) is a reverse PCRprimer for both disease alleles (h) and (i). The forward primertargeting the disease locus has a region of complementarity to thereverse probe targeting the paternal-specific nucleotide variant. Theprocess can be carried out in an emulsion droplet or reaction container(k). FIG. 27 also shows an example of hybridization of primers andtarget nucleic acids in a single cell sequence linkage by ligase chainreaction combined with overlap extension polymerase chain reaction, asapplied to a method for noninvasive prenatal diagnosis, according to oneembodiment of the invention. The process is carried out in an emulsiondroplet or reaction container (k).

Moreover, FIG. 28 shows an example of resulting amplicons produced in asingle cell sequence linkage by ligase chain reaction combined withoverlap extension polymerase chain reaction, as applied to a method fornoninvasive prenatal diagnosis, according to one embodiment of theinvention. FIG. 29 shows hybridization of overlapping complementaryregions of the resulting amplicons, and overlap extension polymerasechain reaction, as applied to a method for noninvasive prenataldiagnosis, according to one embodiment of the invention. FIG. 30illustrates the resulting amplicons that are produced from the overlapextension polymerase chain reaction, as applied to a method fornoninvasive prenatal diagnosis. The end product is a library of “majoramplicons,” or linked loci, which can then be sequenced in bulk.

ii. Methods for Noninvasive Prenatal Molecular Karyotyping

Methods for genetic disease detection are adapted for noninvasiveprenatal molecular karyotyping. Such a method involves the followingsteps: (i) parental genotyping to find paternal-specific polymorphisms;(ii) isolation of single mononuclear cells from maternal blood intoemulsion microdroplets; (iii) amplification of disease andpaternal-specific “linker” loci by a modified LCR/PCR protocol inemulsion microdroplet reactors; (iv) overlap extension amplification ofcomplexes between tens to thousands to hundreds of thousands ofchromosomal probes and linker loci in microdroplet reactors; (v)recovery of linked complexes by emulsion reversal; and (vi) massivelyparallel sequencing. The massively parallel sequencing data are analyzedto quantify instances of linked genotypes. Only microdroplet reactorsthat contain single fetal cells yield linked complexes between thechromosomal probes and the paternal-specific allele. The chromosomalprobes are used to quantify the number of chromosomes or chromosomesegments present in the fetal cells, and, by association, the fetus.Chromosome copy number is quantified by comparing sequence counts froman unknown chromosome to sequence counts from a known referencechromosome within a single experiment, or by looking for allelicimbalance (Johnson et al., 2010 Human Reproduction 25:1066-75). Thismethod is also used to detect a variety of chromosome disorders,including aneuploidy, unbalanced structural chromosome disorders,microdeletions, microinsertions, and other kinds of congenitaldisorders. Examples of disorders of interest include Trisomy 13, Trisomy18, and Trisomy 21.

Example 15 Methods of Noninvasive Cancer Diagnosis

The medical community has long sought noninvasive diagnosis andmonitoring of cancer patients, and there is already an FDA-approvedmethod (CellSearch, Veridex) for quantification of circulating tumorcells for breast and prostate cancer patients. Noninvasive methods fordiagnosis can enable molecular staging of tumors prior to biopsy, whichcan both reduce cost and lead to better clinical outcomes. Aftertreatment, noninvasive methods are used to assess the success of thetreatment regimen without the need for invasive and expensive re-biopsy.There is general consensus among clinicians that noninvasive methods forcharacterization of tumors would greatly benefit patients and increasethe probability of favorable outcomes.

Single cell overlap extension PCR, LCR, padlock probes, and/or RT-PCRare used to specifically analyze only tumor cells in heterogeneous cellpopulations, such as cerebrospinal fluid (CSF) or blood (FIGS. 18-25).Unlike current methods, this approach completely bypasses thecomplexities caused by differences in cell surface markers andmorphology. Such methods are particularly useful in cancers where abiopsy is invasive and expensive, and the treatment decisions, such aspharmacological therapy decisions, would benefit from molecular analysisof the tumor. The technology is used for any kind of tumor or any kindof genetic problem or combination of genetic problems in tumors.

The methods described above in Sections I and II also are used to detecta gene or SNP associated with cancer. Single cell overlap extension PCR,LCR, padlock probes, and/or RT-PCR is used to amplify a first nucleicacid or a second nucleic acid that is associated with cancer. The firsttarget nucleic acid includes a rare somatic mutation and the secondtarget is a gene transcript associated with cancer. Alternatively, onesequence is a molecular barcode and the second sequence is either a raremutation sequence or a gene transcript associated with cancer. In eitheralternative, higher levels of multiplexing produce single-cellexpression patterns for 10, 100, 1000, 10,000 transcripts or even alltranscripts in the cell. Higher levels of multiplexing also can producemutation profiles for entire genes, or many entire genes, or even theentire genome. The rare gene sequence is present in fewer than 5% of thecells, fewer than 1% of the cells, or fewer than 0.1% of the cells. Therare gene sequence results from a genetic mutation. The genetic mutationcan be a somatic mutation. The genetic mutation can be a mutation in agene selected from the group consisting of: epidermal growth factorreceptor (EGFR), phosphatase and tensin homolog (PTEN), tumor protein 53(p53), MutS homolog 2 (MSH2), multiple endocrine neoplasia 1 (MEN1),adenomatous polyposis coli (APC), Fas receptor (FASR), retinoblastomaprotein (Rb1), Janus kinase 2 (JAK2), (ETS)-like transcription factor 1(ELK1), v-ets avian erythroblastosis virus E26 oncogene homolog 1(ETS1), breast cancer 1 (BRCA1), breast cancer 2 (BRCA2), hepatocytegrowth factor receptor (MET), ret protoco-oncogene (RET), V-erb-b2erythroblastic leukemia viral oncogene homolog 2 (HER2), V-Ki-ras2Kirsten rat sarcoma viral oncogene homolog (KRAS), B-cell lymphoma 2(BCL2), V-myc myelocytomatosis viral oncogene homolog (MYC),neurofibromatosis type 2 gene (NF2), v-myb myeloblastosis viral oncogenehomolog (MYB), and mutS homolog 6 (E. coli) (MSH6). Thecancer-associated transcript is a gene selected from the groupconsisting of epidermal cell adhesion molecule (EpCAM), V-erb-b2erythroblastic leukemia viral oncogene homolog 2 (HER2), estrogenreceptor (ER), Signal transducer and activator of transcription 3(STATS), CCAAT-enhancer-binding proteins (C/EBP), prostate-specificantigen (PSA), androgen receptor (AR), progesterone receptor (PR), Jun B(JUNB), Ras-related protein Rab-31 (RAB31), Early growth responseprotein 1 (EGR1), B-cell lymphoma 2 (BCL2), Protein C-ets-1 (ETS1), FBJmurine osteosarcoma viral oncogene homolog (c-Fos), and Insulin-likegrowth factor 1 (IGF-1). Signal transducer and activator oftranscription 2 (STAT2) (Irgon et al., 2010 BMC Cancer 10: 319).

The cancer-associated transcripts can multiplexed to produce a signalfrom 10, 100, 1000, 10,000 transcripts, or all of the transcripts in thecell, which is analyzed by next-generation sequencing to identify amutation. The mutation is associated with cancer. The cancer is selectedfrom the group consisting of lung carcinoma, non-small cell lung cancer,small cell lung cancer, uterine cancer, thyroid cancer, breastcarcinoma, prostate carcinoma, pancreas carcinoma, colon carcinoma,lymphoma, Burkitt lymphoma, Hodgkin lymphoma, myeloid leukemia,leukemia, sarcoma, blastoma, melanoma, seminoma, brain cancer, glioma,glioblastoma, cerebellar astrocytoma, cutaneous T-cell lymphoma, gastriccancer, liver cancer, ependymona, laryngeal cancer, neck cancer, stomachcancer, kidney cancer, pancreatic cancer, bladder cancer, esophagealcancer, testicular cancer, medulloblastoma, vaginal cancer, ovariancancer, cervical cancer, basal cell carcinoma, pituitary adenoma,rhabdomyosarcoma, and Kaposi sarcoma.

The methods in this Example can be applied in an assay using intactmammalian cell mixtures to detect cancer cells. The non-small cell lungcarcinoma cells CRL-5908 (ATCC) is used as a cancer model and Jurkatcells are used as a stand-in for primary lymphocytes. CRL-5908 has anL858R point mutation in EGFR, and expresses EpCAM. Jurkat does notexpress EpCAM (Landolin et al., 2010). Cell mixtures are created at sixCRL-5908: Jurkat ratios between of 0% and 1%. Cells are encapsulatedfrom the mixtures with beads into a lysis mix, and then merged with astream containing a RT-PCR mix using the methods described above. Thecells are diluted such that the cell distribution follows Poissonstatistics with λ=1.5, and ˜44% of the droplets with cells have multiplecells. Using this method, >1 million droplets are generated in each ofsix replicate experiments for each cell mixture. A fast-speed camera isused to obtain bead and cell encapsulation rates. The major ampliconsare purified by gel electrophoresis and sequenced by next-generationsequencing to obtain at least 10 million sequence tags for each library.

Detecting cancer cells in these cell mixtures requires a specialanalytical framework. Sequencing generates counts of mutated EGFR andEpCAM linked to each barcode, and the barcodes are traced back to cells.If each droplet contains a single cell only, then these counts are usedto directly quantify the percentage of CRL-5908 in the cell mixture.However, there may be an arbitrary number of cells encapsulated indroplets according to a Poisson distribution, resulting in many dropletswith multiple cells.

Therefore, for such analysis, an algorithm is used that computes thenumber of cancer cells in a sample given counts of cancer markers suchas mutated EGFR or EpCAM and statistics for cell encapsulation Poissonλ. To test the validity of this algorithm and to estimate the limits ofdetection that encapsulation of multiple cells per droplet imposes, theprocess of encapsulation is simulated, and the ratio of cancer markerexpression in cancer cells to normal cells is determined. A Poissondistribution for the cell encapsulation rate is assumed, log-normallydistributed expression levels over a fixed background, and thesignal-to-noise ratio (SNR) is defined as the ratio of the meanexpression level to the mean background. This simulation indicates a <1%error rate in a scenario where ˜44% of droplets containing cells willhave multiple cells (λ=1.5) and SNR=10.

Example 16 Noninvasive Gene Expression Analysis in GlioblastomaMultiforme

Certain genes are co-expressed specifically only in circulating tumorcells, so linkage of two tumor-specific transcripts in the same cell isa potentially powerful method for detection of circulating tumor cellsin peripheral blood or CSF. The method enables noninvasive molecularstaging of glioblastoma multiforme (GBM). GBM is the most common type ofprimary malignant brain tumor, with an incidence of 16,000 new cases peryear in the United States. After characterization by magnetic resonanceimaging (MRI) and clinical work-up, molecular characterization ofbiopsies is often performed to guide treatment regimens. There isgrowing consensus that distinct molecular categories of tumor should besubjected to distinct targeted treatment regimens (Mischel et al., 2003Cancer Biol Ther 2:242-247). Prior GBM research has indicated that poorprognosis is indicated by coexpression of the genes C/EBPβ and STATS(Carro et al., 2010 Nature 463:318-26). These transcripts are notcoexpressed in normal tissues. However, biopsies of GBM are highlyinvasive and expensive, so there is clinical demand for minimallyinvasive methods for molecular staging.

The method involves the following steps: (i) isolation of mononuclearcells from CSF (Spriggs 1954; Journal of Clinical Pathology 7:122) withemulsion microdroplet technology; (ii) reverse transcriptase polymerasechain reaction targeting C/EBPβ, STAT3, and a linker barcode sequenceunique to each microdroplet; (iii) overlap extension amplification ofcomplexes between C/EBPβ, STAT3, and the linker sequence; (iv) recoveryof linked complexes by emulsion reversal; and (v) digital quantificationof fusion complexes using next-generation sequencing. Only microdropletreactors that contain tumor cells co-expressing C/EBPβ and STAT3 yieldlarge numbers of complete linked complexes. Though next-generationsequencing pools all analytes from all cells, linker barcode sequencesenable the trace back of gene expression to single cells. The finalresult is digital quantification of multiple linked transcripts that aretraced back to millions of single cells analyzed in parallel.

The method also provides cDNA synthesis and PCR in emulsionmicrodroplets without buffer exchange or reagent addition between themolecular steps. Thermostable reverse transcriptase (RT) enzymes areused that withstand temperatures >95° C., such as ThermoScript RT(Lucigen) and GeneAmp Thermostable rTth (Life Technologies). In additionto primer regions targeting C/EBPβ and STAT3 (FIGS. 18-25), three of theprimers in the set include polynucleotide sequences that enableamplification of a fusion complex: (i) the 5′ primer of the C/EBPβ locushas a random 10-20nt sequence with no complementarity to either targetlocus; (ii) the 3′ primer of the C/EBPβ locus has a 10-20nt sequencewith complementarity to the 5′ end of the linker barcodeoligonucleotide; (iii) the 5′ probe of the STAT3 locus hascomplementarity to the 3′ end of the linker. Two more oligonucleotidesact as forward and reverse PCR primers to specifically amplify thelinker barcode oligonucleotide. The “inner” primers of the STAT3 andC/EBPβ loci (i.e., the reverse primer for C/EBPβ and the forward primerfor STAT3) are at limiting concentration, i.e., 0.01 μM for the innerprimers and 0.1 μM for all other primers. This drives amplification ofthe major amplicon preferentially over the minor amplicons.

After emulsion reversal, the major amplicons are subjected to bulksequencing. The barcode is linked to C/EBPβ and STAT3 sequences, and areused to trace back the major amplicons to a single cell (FIGS. 18-25).With trace back of each sequence to an original single cell, it ispossible to tabulate genetic data for each single cell, which thenenables single cell transcript quantification, i.e., single cell geneexpression levels which are translated to a clinically actionablediagnosis.

Example 17 Molecular Karyotyping

Often structural chromosome changes, such as loss of heterozygosity(LOH) or gain of full chromosomes or segments thereof, will lead toprogression of a tumor (Parsons et al., 2008 Science 321:1807-1812).Clinicians often examine the karyotype of a tumor to formulate aprognosis and treatment regimen. The methods outlined above are adaptedto analyze both gene expression and detect chromosome abnormalities forany tumor type in a single multiplexed reaction.

A mutant cancer sequence is linked to probes to determine chromosomecopy number or structural chromosome aberrations. Such a method involvesthe following steps: (i) isolation of single mononuclear cells fromblood into emulsion microdroplets; (ii) amplification of chromosomeprobes and cancer mutation “linker” loci by a modified LCR/PCR protocolin emulsion microdroplet reactors; (iii) overlap extension amplificationof complexes between chromosomal probes and mutant linker loci inmicrodroplet reactors; (iv) recovery of linked complexes by emulsionreversal; and (v) massively parallel sequencing.

The massively-parallel sequencing data is analyzed to quantify instancesof linked genotypes. Only microdroplet reactors that contain cells withcancer mutations yield linked complexes between the chromosomal probesand the cancer-specific sequence. The chromosomal probes are used toquantify the number of chromosomes or chromosome segments present incirculating cancer cells, and, by association, the tumor. Chromosomecopy number is quantified by comparing sequence counts from an unknownchromosome to sequence counts from a known reference chromosome within asingle experiment, or by looking for allelic imbalance (Johnson et al.,2010 Human Reproduction 25:1066-75). This method is also used to detecta variety of chromosome disorders, including aneuploidy, unbalancedstructural chromosome disorders, microdeletions, microinsertions, andother kinds of congenital disorders. The chromosome probes are linked toa barcode sequence rather than a cancer mutation, such that massivelyparallel sequencing measures chromosomal disorders in all of the cellsin the assay rather than just cells that harbor a particular mutation.

Example 18 Somatic Cell Mutations

Often somatic cell mutations, i.e., in tumor promoter genes such as p53,p16, and/or EGFR, contribute to the progression of cancer (Parsons etal., 2008 Science 321:1807-1812). Clinicians often analyze tumors forsuch known somatic cell mutations to formulate a prognosis and treatmentregimen. In particular, somatic cell mutations are often indicative ofprogression to more aggressive stages of a tumor. The methods describedabove are adapted to analyze gene expression, somatic cell mutations,and/or chromosomal changes for any tumor type in multiplexed emulsionmicrodroplet reactions on millions of single cells in parallel. Ifsomatic cell mutations are known, a molecular barcode is not necessarybecause allele-specific LCR or padlock probes are used to specificallyamplify major amplicons only in cells that harbor the somatic cellmutation.

Any combination of gene expression, molecular karyotyping, and somaticcell mutation analysis is carried out in single tumor cells inheterogeneous cell populations. For example, LCR or padlock probes areused to affect allele-specific locus capture and major ampliconamplification only in cells with a particular somatic cell mutation.This method is an alternative to the molecular barcode method describedabove at least at Section B.6), achieving tumor cell-specific geneticanalysis in a highly heterogeneous mixed background of cells. Theallele-specific somatic cell mutation amplification are linked to RNAtranscripts associated with disease outcomes and/or probes forquantification of loss of heterozygosity (LOH) or regional duplicationsin chromosome. The method is used to analyze co-expression of two ormore microRNA sequences in single cells, or co-expression of a microRNAwith another transcript, a methylated DNA sequence, or somatic cellmutation.

Example 19 Methods of Chimeric Cell Population Analysis

Certain applications require multiplexed analysis of cell populationsthat are chimers between two organisms. For example, after hematopoieticstem cell (HSC) transplantation, the host's T and B cells are chimericbetween the host and graft. PCR amplification in a chimeric cellpopulation of a variable genetic locus combined with some kind offunctional genetic locus, such as an RNA transcript, enables analysis ofthe functional genetic locus in an individual-specific manner

The methods described herein are applied to nonmyeloablativehematopoietic stem cell transplantation. Physicians lack powerful toolsfor monitoring patients after nonmyeloablative allogeneic hematopoieticstem cell (HSC) transplants (Pollack et al., 2009 American Journal ofClinical Oncology 32:618-28). After nonmyeloablative transplantation,the host immune system is a chimera between host and graft T cells. Thechimera is a poorly characterized tissue, and chimeric instability isassociated with poor outcome. The balance of donor-recipient immunereconstitution appears to influence a number of transplant immunologicaloutcomes, including graft-versus-tumor effect (GVT), graft-versus-hostdisease (GVHD), and susceptibility to infection. T cells appear to playa major role in mediating each of these processes through adaptiveimmunity and T cell receptor (TCR) antigen recognition. Doctorscurrently lack tools for monitoring host and graft T cell identity afterHSC transplant. Such methods are used to directly monitor GVT, GVHD, andresponse to infections (Kristt et al., 2007 Bone Marrow Transplantation39:255-68).

A method is used to monitor chimeric T cell populations. For T cellchimerism analysis, TCRβ and host- and graft-specific single nucleotidepolymorphisms (SNPs) are linked by overlap extension PCR or overlapextension RT-PCR in single cell microdroplets. This method involves thefollowing steps: (i) genotyping to find SNPs specific to the graft andhost; (ii) post-transplant isolation of single cells from host blood inemulsion microdroplets; (iii) overlap extension PCR amplification offusion complexes between SNPs and TCRβ in microdroplet reactors; and(iv) recovery and sequencing of SNP-TCRβ linkage complexes by emulsionreversal. The result is a library of TCRβ sequences with linkage to hostor graft. The TCRβ sequences are correlated with clinical outcomes overtime.

Similar types of analysis are carried out using LCR, multi-probecircularization, or padlock probes, or any combination thereof. Also,other types of variant sequences, such as STRs, are also useful toindicate cell source in a chimeric population of cells. The T cellchimerism analysis is adaptable to applications such as B cell analysisor any other subpopulation of mononuclear cells in blood. Additionally,the method is combinable with functional T cell sequencing to indicatethe immune activity of particular T cell clones.

There are many applications for chimeric cell population analysisoutside of the field of medicine. For example, an investigator maycreate chimeric organisms, such as fruit flies, mice, or rats, which arechimers between multiple individuals with different genetic backgrounds,or even between multiple species. Chimeric cell populations for RNAtranscripts, DNA methylation, somatic cell mutations, presence of arecombinant gene, or a variable DNA region are also capable of analysiswith this method. Thus methods for analysis of chimeric T and B cellpopulations are adapted to other organisms and other kinds of cellpopulations. Additionally, such methods are used for allogeneic orautologous cellular therapeutics. Currently physicians lack powerfultools for monitoring patients after immune cells have been introducedeither from a donor or as previously harvested from the patient. Tcells, B cells, or NK cells are monitored to establish characteristicsand efficacy of therapy.

Example 20 Methods for Gene Regulatory Sequence Analysis

Variants in regulatory DNA have an impact on expression levels of RNAtranscripts (Brown et al., 2007 Science 317:1557-60). Functional screensof regulatory variants are time-consuming and expensive. In oneexemplary method, the method includes mutagenizing cells, capturingsingle cells in aqueous-in-oil microdroplets, and then fusing anamplified putative regulatory locus with RNA transcripts from the nearbygene. In this way, mutations in regulatory sequences could be linkedwith gene expression levels.

Often an investigator wishes to understand how genomic nucleic acidsequences impact expression of transcripts. Because many nucleotidesimpact gene expression, these experiments are tedious. Ideally, aninvestigator would want to analyze quantitative gene expression at thesingle cell level as a function of mutagenized regulatory sequences.Suspected regulatory sequences are mutagenized to create a library ofvariable regulatory sequences. Then, a combination of overlap extensionPCR and overlap extension RT-PCR in single cell emulsion microdropletsis used to link regulatory DNA sequence to RNA transcript levels. Inthis way, the effect of regulatory sequence mutagenesis on RNAtranscript levels is measured in single cells.

Example 21 Methods for Molecular Haplotyping

Many kinds of genetic analysis, such as PGD or whole genome associationstudies, benefit from haplotype linkage of several genetic loci in DNAderived from a single sperm cell. In one exemplary method, single spermcells are captured in aqueous-in-oil microdroplets, and then severalvariable genetic loci are fused in the cells, such as SNPs or STRs. Thisenables massively parallel molecular phasing.

An method for phasing of two loci is provided. Haplotypes millions ofsingle sperm are analyzed in parallel. The method involves the followingsteps: (i) isolation of single sperm cells using emulsion microdroplettechnology; (ii) amplification of two genetic variants by PCR inmicrodroplet reactors; (iii) overlap extension PCR amplification offusion complexes between the variants in microdroplet reactors; and (iv)recovery of linked complexes by emulsion reversal. The result is alibrary of phased haplotypes, which are then sequenced usingnext-generation sequencing.

An alternate method for phasing of multiple loci is provided in thisparagraph. In some cases, phasing of only two loci is not adequate forimprovement of whole genome association studies or other kinds ofanalysis. In such situations, molecular methods such as LCR or padlockprobes, which enable higher probe multiplexing, are used as analternative. Additionally, a variety of PCR primer pairs are affixed tobeads, such that thousands of PCR primer pairs are distributed toemulsion microdroplets that contain a single bead and a single spermcell.

Example 22 Methods of Detecting Directed Molecular Evolution

Some kinds of industrial applications require improved enzymes and/orbiological strains to optimize engineered biosystems. For example,enzymes that degrade a particular kind of industrial waste might not befound in nature, but in vitro evolution of existing enzymes might resultin an optimized enzyme. Many such processes benefit from moleculargenetic analysis of multiple loci in millions of single cells analyzedin parallel.

Yeast Evolution

Industrial in vitro evolution often involves mutagenesis of cellsfollowed by growth in selective media. In an exemplary method, yeastcells are mutagenized and grown on special media containing xylose asthe primary food source. The single yeast cells are captured inaqueous-in-oil microdroplets, and then several metabolic pathway genesare sequenced. At least one company (Microbiogen, Sydney, AUS) isdeveloping yeast strains for growth on xylose, but is using slow,traditional screening methods.

Other Applications

Many groups are currently researching methods for improving naturalstrains of algae and bacteria for the purpose of biofuel production. Themethods for linked genotyping and/or single cell gene expressionanalysis are used to enable in vitro evolution of these organisms forthe purpose of biofuel or other kinds of energy production.

Example 23 Other Applications and Derivative Methods

Agriculture

All of the clinical methods described above (e.g., T cell sequencing andB cell sequencing) are applicable to animals. These animals include, butare not limited to, cows, pigs, chickens, or salmon, etc. In particular,livestock and other agricultural animals suffer from infectious disease,which results in considerable economic hardships. The methods describedherein are adaptable to improve monitoring and detection of infectiousdisease in an agricultural setting.

Metagenomics

Metagenomics is a method of studying genetic diversity in ecosystems inwhich environmental samples are directly sequenced. In an exemplarymethod, cells such as algae in environmental samples such as seawaterare separated into single cell emulsion microdroplets, and then analyzedfor at least two genetic loci. For example, an investigator may beinterested to find a particular species of algae that expresses aparticular form of chlorophyll and belongs to a particular algalspecies. Genotyping by LCR is used to amplify major amplicons only algalcells from a particular species that harbor that particular form ofchlorophyll. One skilled in the art can also appreciate that such amethod are also useful to sample chlorophyll diversity in a particularclass, species, or genus of algae by linking species-specific LCR orpadlock probes with PCR amplification of chlorophyll exons. Algae andchlorophyll are only one specific example; the cells are from anyspecies, and there are many kinds of target genetic loci, including RNAtranscripts, genomic variants, and mitochondrial DNA.

Detection of DNA Methylation

DNA methylation is a type of epigenetic modifier that helps cellscontrol RNA transcription and other cellular processes (Brunner et al.,2009 Genome Research 19:1044-56). For example, blood lymphocytes cansuffer aberrant DNA methylation, leading to liquid tumors. The methodsdescribed above are useful for analyzing DNA methylation in single cells(e.g., multiple DNA methylation loci in single cells, or at least oneDNA methylation locus with an RNA transcript target or DNA sequencetarget). DNA methylation is analyzed by methylation-specific restrictionenzymes, bisulfite conversion, or precipitation withanti-methylcytosine. Most of these analyses would require multipleinputs of reaction buffers if using a microfluidic chip to createemulsion microdroplets. For example, performing bisulfite conversionrequires a buffer that is inappropriate for PCR, LCR, RT-PCR, or padlockprobes. In an exemplary method, single cells are encapsulated inemulsion microdroplets using a standard bisulfite conversion buffer.Then, after bisulfite conversion, the microdroplets are merged with asecond aqueous buffer. This second buffer dilutes the bisulfiteconversion buffer, enabling PCR, LCR, RT-PCR, or padlock probe methods.Similar approaches are useful for anti-methylcytosine or methylationsensitive restriction digestion.

Chromatin Immunoprecipitation

Chromatin immunoprecipitation is a method in which DNA is crosslinked toproteins in cell nuclei (Johnson et al., 2007 Science 316:1497-502). Anantibody directed against a DNA binding protein of interest is then usedto specifically precipitate DNA-protein complexes, and then the DNA issequenced or analyzed with a DNA microarray. The molecular linkagemethods described above are used to analyze multiple DNA-protein bindingloci in single cells, or at least one DNA-protein binding locus with anRNA transcript target or DNA sequence target. Most of these analysesrequire multiple inputs of reaction buffers if using a microfluidic chipto create emulsion microdroplets. For example, performing chromatinimmunoprecipitation requires a buffer that is inappropriate for PCR,LCR, RT-PCR, or padlock probes. In an exemplary method, single cells areencapsulated in emulsion microdroplets using a standardimmunoprecipitation buffer. Then, after precipitation, the microdropletsare merged with a second aqueous buffer. This second buffer dilutes theprecipitation buffer, enabling PCR, LCR, RT-PCR, or padlock probemethods.

While the invention has been particularly shown and described withreference to a preferred s and various alternate embodiments, it will beunderstood by persons skilled in the relevant art that various changesin form and details can be made therein without departing from thespirit and scope of the invention.

All references, issued patents and patent applications cited within thebody of the instant specification are hereby incorporated by referencein their entirety, for all purposes.

TABLE 2 INFORMAL SEQUENCE LISTING SEQ ID OVERLAP NO NAMEGENOME TARGETING REGION SEQUENCE DESCRIPTION SEQ ID TRBV2.FTCAAATTTCACTCTGAAGATCCGGTCCACAA outer forward NO: 1 SEQ ID TRBV3-GCTCACTTAAATCTTCACATCAATTCCCTGG outer forward NO: 2 1.F SEQ ID TRBV4-CTTAAACCTTCACCTACACGCCCTGC outer forward NO: 3 1.F SEQ ID TRBV4-CTTATTCCTTCACCTACACACCCTGC outer forward NO: 4 2_4-3.F SEQ ID TRBV5-GCTCTGAGATGAATGTGAGCACCTTG outer forward NO: 5 1.F SEQ ID TRBV5-GCTCTGAGATGAATGTGAGTGCCTTG outer forward NO: 6 3.F SEQ ID TRBV_5-GCTCTGAGCTGAATGTGAACGCCTTG outer forward NO: 7 4_5-5_5- 6_5-7_5- 8.FSEQ ID TRBV6- TCGCTCAGGCTGGAGTCGGCTG outer forward NO: 8 1.F SEQ IDTRBV6- GCTGGGGTTGGAGTCGGCTG outer forward NO: 9 2_6-3.F SEQ ID TRBV6-CCCTCACGTTGGCGTCTGCTG outer forward NO: 10 4.F SEQ ID TRBV6-GCTCAGGCTGCTGTCGGCTG outer forward NO: 11 5.F SEQ ID TRBV6-CGCTCAGGCTGGAGTTGGCTG outer forward NO: 12 6.F SEQ ID TRBV6-CCCCTCAAGCTGGAGTCAGCTG outer forward NO: 13 7.F SEQ ID TRBV6-CACTCAGGCTGGTGTCGGCTG outer forward NO: 14 8.F SEQ ID TRBV6-CGCTCAGGCTGGAGTCAGCTG outer forward NO: 15 9.F SEQ ID TRBV7-CCACTCTGAAGTTCCAGCGCACAC outer forward NO: 16 1.F SEQ ID TRBV7-CACTCTGACGATCCAGCGCACAC outer forward NO: 17 2.F SEQ ID TRBV7-CTCTACTCTGAAGATCCAGCGCACAG outer forward NO: 18 3.F SEQ ID TRBV7-CCACTCTGAAGATCCAGCGCACAG outer forward NO: 19 4.F SEQ ID TRBV7-CACTCTGACGATCCAGCGCACAG outer forward NO: 20 6.F SEQ ID TRBV7-CCACTCTGACGATTCAGCGCACAG outer forward NO: 21 7.F SEQ ID TRBV7-CCACTCTGAAGATCCAGCGCACAC outer forward NO: 22 8.F SEQ ID TRBV7-CACCTTGGAGATCCAGCGCACAG outer forward NO: 23 9.F SEQ ID TRBV9.FGCACTCTGAACTAAACCTGAGCTCTCTG outer forward NO: 24 SEQ ID TRBV10-CCCCTCACTCTGGAGTCTGCTG outer forward NO: 25 1.F SEQ ID TRBV10-CCCCCTCACTCTGGAGTCAGCTA outer forward NO: 26 2.F SEQ ID TRBV10-CCTCCTCACTCTGGAGTCCGCTA outer forward NO: 27 3.F SEQ ID TRBV11-CCACTCTCAAGATCCAGCCTGCAG outer forward NO: 28 1_11-3.F SEQ ID TRBV11-CTCCACTCTCAAGATCCAGCCTGCAA outer forward NO: 29 2.F SEQ ID TRBV12-CCACTCTGAAGATCCAGCCCTCAG outer forward NO: 30 3_12-4_12- 5.F SEQ IDTRBV13.F CATTCTGAACTGAACATGAGCTCCTTGG outer forward NO: 31 SEQ IDTRBV14.F CTACTCTGAAGGTGCAGCCTGCAG outer forward NO: 32 SEQ ID TRBV15.FGATAACTTCCAATCCAGGAGGCCGAACA outer forward NO: 33 SEQ ID TRBV16.FCTGTAGCCTTGAGATCCAGGCTACGA outer forward NO: 34 SEQ ID TRBV17.FCTTCCACGCTGAAGATCCATCCCG outer forward NO: 35 SEQ ID TRBV18.FGCATCCTGAGGATCCAGCAGGTAG outer forward NO: 36 SEQ ID TRBV19.FCCTCTCACTGTGACATCGGCCC outer forward NO: 37 SEQ ID TRBV20-CTTGTCCACTCTGACAGTGACCAGTG outer forward NO: 38 1.F SEQ ID TRBV23-CAGCCTGGCAATCCTGTCCTCAG outer forward NO: 39 1.F SEQ ID  TRBV24-CTCCCTGTCCCTAGAGTCTGCCAT outer forward NO: 40 1.F SEQ ID TRBV25-CCCTGACCCTGGAGTCTGCCA outer forward NO: 41 1.F SEQ ID TRBV27.FCCCTGATCCTGGAGTCGCCCA outer forward NO: 42 SEQ ID TRBV28.FCTCCCTGATTCTGGAGTCCGCCA outer forward NO: 43 SEQ ID TRBV29-CTAACATTCTCAACTCTGACTGTGAGCAACA outer forward NO: 44 1.F SEQ ID TRBV30.FCGGCAGTTCATCCTGAGTTCTAAGAAGC outer forward NO: 45 SEQ ID TRBJ1-1.RTTACCTACAACTGTGAGTCTGGTGCCTTGTC GCTCATCTGGC inner reverse NOS 46 CAAAATAATTCTCCT and 74 SEQ ID TRBJ1-2.R ACCTACAACGGTTAACCTGGTCCCCGAACCGGCTCATCTGGC inner reverse NOS 47 AA ATAATTCTCCT and 74 SEQ ID TRBJ1-3.RACCTACAACAGTGAGCCAACTTCCCTCTCCA GCTCATCTGGC inner reverse NOS 48 AAATAATTCTCCT and 74 SEQ ID TRBJ1-4.R CCAAGACAGAGAGCTGGGTTCCACTGCCAAAGCTCATCTGGC inner reverse NOS 49 ATAATTCTCCT and 74 SEQ ID TRBJ1-6.RCTGTCACAGTGAGCCTGGTCCCGTTCCCAAA GCTCATCTGGC inner reverse NOS 50ATAATTCTCCT and 74 SEQ ID TRBJ2-1.R CGGTGAGCCGTGTCCCTGGCCCGAAGCTCATCTGGC inner reverse NOS 51 ATAATTCTCCT and 74 SEQ ID TRBJ2-2.RCCAGTACGGTCAGCCTAGAGCCTTCTCCAAA GCTCATCTGGC inner reverse NOS 52ATAATTCTCCT and 74 SEQ ID TRBJ2-3.R ACTGTCAGCCGGGTGCCTGGGCCAAAGCTCATCTGGC inner reverse NOS 53 ATAATTCTCCT and 74 SEQ ID TRBJ2-4.RAGAGCCGGGTCCCGGCGCCGAA GCTCATCTGGC inner reverse NOS 54 ATAATTCTCCTand 74 SEQ ID TRBJ2-5.R GGAGCCGCGTGCCTGGCCCGAA GCTCATCTGGC inner reverseNOS 55 ATAATTCTCCT and 74 SEQ ID TRBJ2-6.R GTCAGCCTGCTGCCGGCCCCGAAGCTCATCTGGC inner reverse NOS 56 ATAATTCTCCT and 74 SEQ ID TRBJ2-7.RGTGAGCCTGGTGCCCGGCCCGAA GCTCATCTGGC inner reverse NOS 57 ATAATTCTCCTand 74 SEQ ID IL2.F TCACCAGGATGCTCACATTTAAGT AGGAGAATTAT inner forwardNOS 58 GCCAGATGAGC and 74 SEQ ID IL2.F GAGGTTTGAGTTCTTCTTCTAGACACTGAouter reverse NO: 59 and 74 SEQ ID IL4.F CCACGGACACAAGTGCGATAAGGAGAATTAT inner forward NOS 60 GCCAGATGAGC and 74 SEQ ID IL4.RCCCTGCAGAAGGTTTCCTTCT outer reverse NO:61 and 74 SEQ ID INFG.FTCAGCTCTGCATCGTTTTGG AGGAGAATTAT inner forward NOS 62 GCCAGATGAGC and 75SEQ ID INFG.R GTTCCATTATCCGCTACATCTGAA outer reverse NO: 63 SEQ IDTNFA.F GCCCAGGCAGTCAGATCATC AGGAGAATTAT inner forward NOS 64 GCCAGATGAGCand 75 SEQ ID TNFA.R GGGTTTGCTACAACATGGGCT outer reverse NO: 65 SEQ IDFOXP3.F AACAGCACATTCCCAGAGTTCCT AGGAGAATTAT inner forward NOS 66GCCAGATGAGC and 75 SEQ ID FOXP3.R CATTGAGTGTCCGCTGCTTCT outer reverseNO: 67 SEQ ID TBX21.F GTCCAACAATGTGACCCAGAT AGGAGAATTAT inner forwardNOS 68 GCCAGATGAGC and 75 SEQ ID TBX21.R GCTGGTACTTATGGAGGGACTGouter reverse NO: 69 SEQ ID TBX21.F AGCTGACTCACGCCGTCC AGGAGAATTATinner forward NOS 70 GCCAGATGAGC and 75 SEQ ID TBX21.FCACAGAAACCCTCGCACAAGCC outer reverse NO: 71 SEQ ID IL2.FCTGGAATAGCCAATACTGATTACCTG AGGAGAATTAT inner forward NOS 72 GCCAGATGAGCand 75 SEQ ID IL2.R CATGAATTTTATACCTTAGGAGACGG outer reverse NO: 73

1. A method for analyzing at least two nucleic acid sequences in asingle cell contained within a population of at least 10,000 cells,comprising: isolating each of a plurality of single cells from apopulation of at least 10,000 cells in an emulsion microdroplet or areaction container, introducing a unique barcode sequence comprising atleast six nucleotides into each of the plurality of single cells,wherein each barcode sequence is selected from a pool of barcodesequences with greater than 1000-fold diversity in sequence; for each ofthe plurality of single cells, providing at least one set of nucleicacid probes, the set comprising a first probe comprising a sequence thatis complementary to a nucleic acid sequence that is located at the 5′end of the barcode sequence, a second probe comprising a sequence thatis complementary to a nucleic acid sequence that is located at the 3′end of the barcode sequence and a second region of sequence that iscomplementary to a non-human, exogenous sequence, a third probecomprising a sequence that comprises the non-human, exogenous sequenceand a sequence that is complementary to a first subsequence of a secondtarget nucleic acid sequence, and a fourth probe comprising a sequencethat is complementary to a second subsequence of the second targetnucleic acid sequence; amplifying the first and second nucleic acidsequences independently, wherein the first target nucleic acid sequenceis amplified using the first probe and the second probe, and wherein thesecond target nucleic acid sequence is amplified using the third probeand the fourth probe; hybridizing the exogenous sequence to itscomplement; amplifying the first target nucleic acid sequence, thesecond target nucleic acid sequence, and the exogenous sequence usingthe first and fourth probes; performing bulk sequencing of the fusedcomplexes; and identifying a single cell for each of the fused complexesbased on the barcode sequence.
 2. The method of claim 1, wherein thebarcode sequence is affixed to a bead or a solid surface.
 3. The methodof claim 2, wherein the bead or the solid surface is isolated in theemulsion microdroplet or the reaction container.
 4. The method of claim3, wherein introducing a unique barcode sequence comprises fusing theemulsion microdroplet or a reaction container comprising the single cellwith the emulsion microdroplet or a reaction container comprising thebarcode sequence affixed to the bead or the solid surface.
 5. The methodof claim 1, wherein the second target nucleic acid sequence iscomplementary to an RNA sequence.
 6. The method of claim 1, wherein thesecond target nucleic acid sequence is complementary to a DNA sequence.7. The method of claim 1, wherein amplifying comprises performing apolymerase chain reaction.
 8. The method of claim 1, wherein amplifyingcomprises performing a ligase chain reaction.
 9. The method of claim 1,wherein amplifying comprises performing by ligase chain reactionfollowed by polymerase chain reaction.
 10. The method of claim 1,wherein the single cell is contained within a population of at least25,000 cells.
 11. The method of claim 10, wherein the single cell iscontained within a population of at least 50,000 cells.
 12. The methodof claim 11, wherein the single cell is contained within a population ofat least 75,000 cells.
 13. The method of claim 12, wherein the singlecell is contained within a population of at least 100,000 cells.
 14. Themethod of claim 1, further comprising quantifying the fused complexes.15. The method of claim 1, wherein the fused complexes are circular. 16.The method of claim 1, further comprising: providing N sets of nucleicacid probes, wherein each of the N sets comprise an I₁ probe comprisinga sequence that is complementary a first subsequence of a barcodesequence, an I₂ probe comprising a sequence that is complementary to asecond subsequence of the barcode sequence and a second sequence that iscomplementary to an I exogenous sequence, an I₃ probe comprising the Iexogenous sequence and a sequence that is complementary to an I_(b)target nucleic acid first subsequence, and an I₄ probe comprising asequence that is complementary to an I_(b) target nucleic acid secondsubsequence, wherein I ranges from 1 to N; isolating the single cellswith the N sets of nucleic acid probes; amplifying for all values of I,the barcode sequence and the I_(b) target nucleic acid sequencesindependently, wherein the barcode sequence is amplified using the I₁probe and the I₂ probe and the I_(b) target nucleic acid sequence isamplified using the I₃ probe and the I₄ probe; hybridizing the Iexogenous sequence to its complement; amplifying for each I, the barcodesequence, the I_(b) target sequence and the I exogenous sequence usingthe I₁ and L₄ probes, thereby generating N fused complexes; andperforming a bulk sequencing reaction to generate sequence informationfor at least 100,000 fused complexes from at least 10,000 cells withinthe population of cells, wherein the sequence information is sufficientto co-localize the barcode sequence and the I_(b) target nucleic acidsequence to a single cell from the population of at least 10,000 cells.17. The method of claim 16, wherein N is less than or equal to
 10. 18.The method of claim 16, wherein N is less than or equal to
 100. 19. Themethod of claim 16, wherein N is less than or equal to
 1000. 20. Themethod of claim 16, wherein N is less than or equal to 10,000.
 21. Themethod of claim 16, wherein N is less than or equal to 100,000.
 22. Themethod of claim 16, wherein N represents all of the polyadenylatedtranscripts in a cell.
 23. The method of claim 16, wherein the barcodesequence is the same sequence for all N.
 24. A method for introducingunique barcode sequences into reaction containers or emulsionmicrodroplets, comprising: providing a pool of unique barcode sequences,wherein each barcode sequence is linked to a selection resistance gene;providing a population of single cells; transfecting the population ofsingle cells with the pool of unique barcode sequences; selecting cellscomprising a unique barcode sequence and the selection resistance gene;and isolating each of the selected cells into reaction containers oremulsion microdroplets.
 25. The method of claim 24, wherein theselection resistance gene comprises a gene that encodes resistance togentamycin, neomycin, hygromycin, or puromycin.
 26. A method foranalyzing at least two nucleic acid sequences in a single cell containedwithin a population of at least 10,000 cells, comprising: providing afirst set of nucleic acid probes, the first set comprising a first probecomprising a sequence that is complementary to a first target nucleicacid subsequence, a second probe comprising a sequence that iscomplementary to a second subsequence of the first target nucleic acidand a second sequence that is complementary to an exogenous sequence, athird probe comprising the exogenous sequence and a sequence that iscomplementary to a first subsequence of a second target nucleic acid,and a fourth probe comprising a sequence that is complementary to asecond subsequence of the second target nucleic acid sequence, whereinthe first target nucleic acid or the second target nucleic acidcomprises an endogenous sequence; isolating the single cells with atleast one set of nucleic acid probes; amplifying the first and secondtarget nucleic acid sequences independently, wherein the first targetnucleic acid sequence is amplified using the first probe and the secondprobe, and wherein the second target nucleic acid sequence is amplifiedusing the third probe and the fourth probe; hybridizing the exogenoussequence to its complement; amplifying the first target nucleic acidsequence, the second target nucleic acid sequence, and the exogenoussequence using the first and fourth probes, thereby generating a fusedcomplex; and performing a bulk sequencing reaction to generate sequenceinformation for at least 100,000 fused complexes from at least 10,000cells within the population of cells, wherein the sequence informationis sufficient to co-localize the first target nucleic acid sequence andthe second target nucleic acid sequence to a single cell from thepopulation of at least 10,000 cells.